Novel serine protease reagents and methods useful for detecting and treating diseases of the prostate

ABSTRACT

A set of contiguous and partially overlapping RNA sequences and polypeptides encoded thereby, designated as PS133 and transcribed from prostate tissue is described. One polypeptide of the present invention is a member of the human serine protease family. These sequences are useful for the detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining the predisposition of an individual to diseases and conditions of the prostate, such as prostate cancer. Also provided are antibodies which specifically bind to PS133-encoded polypeptide or protein, and agonists or inhibitors which prevent action of the tissue-specific PS133 polypeptide, which molecules are useful for the therapeutic treatment of prostate diseases, tumors or metastases.

BACKGROUND OF THE INVENTION

[0001] The invention relates generally to detecting diseases of theprostate, and more particularly, relates to reagents such aspolynucleotide sequences and the polypeptide sequences encoded thereby,as well as methods which utilize these sequences, which are useful fordetecting, diagnosing, staging, monitoring, prognosticating, preventingor treating, or determining predisposition to diseases or conditions ofthe prostate such as prostate cancer. One polypeptide of the presentinvention is a serine protease.

[0002] Prostate cancer is the most common form of cancer occurring inmales in the United States, with projections of 334,500 new casesdiagnosed and 41,800 related deaths predicted to occur during 1997(American Cancer Society). Prostate cancer also has shown the largestincrease in incidence as compared to other types of cancer, increasing142% from 1992 to 1996.

[0003] Procedures used for detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, or determining predispositionto diseases or conditions of the prostate such as prostate cancer are ofcritical importance to the outcome of the patient. For example, patientsdiagnosed with localized prostate cancer have greater than a 90%five-year survival rate compared to a rate of 25 to 31% for patientsdiagnosed with distant metastasis. (American Cancer Society statistics).A diagnostic procedure for early detection of prostate cancer should,therefore, specifically detect this disease and be capable of detectingthe presence of prostate cancer before symptoms appear.

[0004] Such procedures could include assays based upon the appearance ofvarious disease markers in test samples such as blood, plasma, serum, orurine obtained by minimally invasive procedures which are detectable byimmunological methods. These procedures would provide information to aidthe physician in managing the patient with disease of the prostate andat low cost to the patient. Markers such as the prostate specificantigen (PSA) exist and are used clinically for screening patients forprostate cancer. Elevated levels of PSA protein in serum can be used asa marker in the early detection of prostate cancer in asymptomatic men.G. E. Hanks, et al., In: Cancer: Principles and Practice of Oncology,Vol. 1, Fourth Edition, pp. 1073-1113, Philadelphia, Pa.: J. B.Lippincott Co. (1993.). PSA normally is secreted by the prostate at highlevels into the seminal fluid, but is present in very low levels in theblood of men with normal prostates. However, in patients with diseasesof the prostate including benign prostatic hyperplasia (BPH) andadenocarcinoma of the prostate, the level of PSA can be markedlyelevated in the blood and thus be useful as an indicator of prostatedisease. PSA, however, cannot differentiate between BPH and prostatecancer, which reduces its specificity as a marker for prostate cancer.M. K. Schwartz, et al., In: Cancer: Principles and Practice of Oncology,Vol. 1, Fourth Edition, pp. 531-542, Philadelphia, Pa.: J. B. LippincottCo. 1993. New markers which are more specific for prostate cancer thuswould be beneficial in the initial detection of this disease.

[0005] A critical step in managing patients with prostate cancer is thepresurgical staging of the cancer to provide prognostic value andcriteria for designing optimal therapy. Improved procedures foraccurately staging prostate cancer prior to surgery are needed. Onestudy demonstrated that current methods of staging prostate cancer priorto surgery were incorrect approximately fifty percent (50%) of the time.F. Labrie, et al., Urology 44 (Symposium Issue): 29-37 (1994). Prostatecancer management also could be improved by utilizing new markers foundin an inappropriate body compartment. Such markers could be mRNA orprotein markers expressed by cells originating from the primary prostatetumor but residing in blood, bone marrow or lymph nodes and could besensitive indicators for metastasis to these distal organs. For example,in patients with metastatic prostate cancer, PSA protein has beendetected by immunohistochemical techniques in bone marrow, and PSA mRNAhas been detected by RT-PCR in cells of blood, lymph nodes and bonemarrow. K. Pantel, et al., Onkologie 18: 394-401 (1995).

[0006] New markers which could predict the biologic behavior of earlyprostate cancers would also be of significant value. Early prostatecancers that threaten or will threaten the life of the patient are moreclinically important than those that do not or will not be a threat. G.E. Hanks, supra. A need therefore exists for new markers which candifferentiate between the clinically important and unimportant prostatecancers. Such markers would allow the clinician to accurately identifyand effectively treat early cancers localized to the prostate whichcould otherwise metastasize and kill the patient. Further, if one couldshow that such a marker characteristic of aggressive cancer was absent,the patient could be spared expensive and non-beneficial treatment.

[0007] It also would be beneficial to find a prostate associated markerwhich is more sensitive in detecting recurrence of prostate cancer thanPSA and which is not affected by androgens. To date, PSA has proven tobe the most sensitive marker for detecting recurrent disease. However,in some cases tumor progression occurs without PSA elevation due tohormonal therapy utilized for treating the cancer. Although the decreasein androgen results in a concomitant decrease in PSA, it does notnecessarily reflect a decrease in tumor metastasis. This complication isthe result of androgen-stimulated PSA expression. Part of the decline inPSA observed after androgen ablation is due not to tumor cell death butto diminished PSA expression. G. E. Hanks, supra.

[0008] PSA is a member of the human kallikrein gene family which is afamily of serine proteases. A serine protease is a class of proteinwhose function is to accelerate proteolysis of a substrate protein.Proteolytic processes are thought to be the critical point in tumorinvasion and metastasis, and are performed mainly by matrixmetalloproteinases (MMPs) and serine proteases or proteinases. In somecases, apoptosis, or programmed cell death, may be prevented by serineprotease inhibitors. Suzuki et al., Exp. Cell Res. 233 (1):48-55 (1997).

[0009] It therefore would be advantageous to provide specific methodsand reagents for detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, or determining predispositionto diseases and conditions of the prostate, or to indicate possiblepredisposition to these conditions. Such methods would include assayinga test sample for products of a gene which are overexpressed in prostatediseases and conditions such as cancer. Such methods may also includeassaying a test sample for products of a gene alteration associated withprostate disease or condition. Such methods may further include assayinga test sample for products of a gene whose distribution among thevarious tissues and compartments of the body have been altered by aprostate-associated disease or condition such as cancer. Useful reagentsinclude polynucleotides, or fragments thereof, which may be used indiagnostic methods such as reverse transcriptase-polymerase chainreaction (RT-PCR), PCR, or hybridization assays of mRNA extracted frombiopsied tissue, blood or other test samples. Other useful reagentsinclude polypeptides or proteins which are the translation products ofsuch mRNAs, and antibodies directed against these polypeptides orproteins. Drug treatment or gene therapy for diseases or conditions ofthe prostate can then be based on these identified gene sequences ortheir expressed proteins and efficacy of any particular therapy can bemonitored. Furthermore, it would be advantageous to have availablealternative non-surgical diagnostic methods capable of detecting earlystage prostate disease such as cancer.

SUMMARY OF THE INVENTION

[0010] The present invention provides a method of detecting a targetPS133 polynucleotide in a test sample which comprises contacting thetest sample with at least one PS133-specific polynucleotide anddetecting the presence of the target PS8133 polynucleotide in the testsample. The PS133-specific polynucleotide has at least 50% identity witha polynucleotide selected from the group consisting of SEQUENCE ID NOS1-8, and fragments or complements thereof. Also, the PS133-specificpolynucleotide may be attached to a solid phase prior to performing themethod.

[0011] The present invention also provides a method for detecting PS133mRNA in a test sample, which comprises performing reverse transcription(RT) with at least one primer in order to produce cDNA, amplifying thecDNA so obtained using PS133 oligonucleotides as sense and antisenseprimers to obtain PS133 amplicon, and detecting the presence of thePS133 amplicon as an indication of the presence of PS133 mRNA in thetest sample, wherein the PS133 oligonucleotides have at least 50%identity to a sequence selected from the group consisting of SEQUENCE IDNOS 1-8, and fragments or complements thereof. Amplification can beperformed by the polymerase chain reaction. Also, the test sample can bereacted with a solid phase prior to performing the method, prior toamplification or prior to detection. This reaction can be a direct or anindirect reaction. Further, the detection step can comprise utilizing adetectable label capable of generating a measurable signal. Thedetectable label can be attached to a solid phase.

[0012] The present invention further provides a method of detecting atarget PS133 polynucleotide in a test sample suspected of containingtarget PS133 polynucleotides, which comprises (a) contacting the testsample with at least one PS133 oligonucleotide as a sense primer and atleast one PS133 oligonucleotide as an anti-sense primer, and amplifyingsame to obtain a first stage reaction product; (b) contacting the firststage reaction product with at least one other PS133 oligonucleotide toobtain a second stage reaction product, with the proviso that the otherPS133 oligonucleotide is located 3′ to the PS133 oligonucleotidesutilized in step (a) and is complementary to the first stage reactionproduct; and (c) detecting the second stage reaction product as anindication of the presence of a target PS133 polynucleotide in the testsample. The PS133 oligonucleotides selected as reagents in the methodhave at least 50% identity to a sequence selected from the groupconsisting of SEQUENCE ID NOS 1-8, and fragments or complements thereof.Amplification may be performed by the polymerase chain reaction. Thetest sample can be reacted either directly or indirectly with a solidphase prior to performing the method, or prior to amplification, orprior to detection. The detection step also comprises utilizing adetectable label capable of generating a measurable signal; further, thedetectable label can be attached to a solid phase. Test kits useful fordetecting target PS133 polynucleotides in a test sample are alsoprovided which comprise a container containing at least onePS133-specific polynucleotide selected from the group consisting ofSEQUENCE ID NOS 1-8, and fragments or complements thereof. These testkits further comprise containers with tools useful for collecting testsamples (such as, for example, blood, urine, saliva and stool). Suchtools include lancets and absorbent paper or cloth for collecting andstabilizing blood; swabs for collecting and stabilizing saliva; and cupsfor collecting and stabilizing urine or stool samples. Collectionmaterials, such as papers, cloths, swabs, cups and the like, mayoptionally be treated to avoid denaturation or irreversible adsorptionof the sample. The collection materials also may be treated with orcontain preservatives, stabilizers or antimicrobial agents to helpmaintain the integrity of the specimens.

[0013] The present invention provides a purified polynucleotide orfragment thereof derived from a PS133 gene. The purified polynucleotideis capable of selectively hybridizing to the nucleic acid of the PS133gene, or a complement thereof. The polynucleotide has at least 50%identity to a polynucleotide selected from the group consisting ofSEQUENCE ID NOS 1-5, SEQUENCE ID NO 8, and fragments or complementsthereof, or at least 50% identity with SEQUENCE ID NO 7. Further, thepurified polynucleotide can be produced by recombinant and/or synthetictechniques. The purified recombinant polynucleotide can be containedwithin a recombinant vector. The invention further comprises a host celltransfected with said vector.

[0014] The present invention further provides a recombinant expressionsystem comprising a nucleic acid sequence that includes an open readingframe derived from PS133. The nucleic acid sequence has at least 50%identity with a sequence selected from the group consisting of SEQUENCEID NOS 1-8, and fragments or complements thereof. The nucleic acidsequence is operably linked to a control sequence compatible with adesired host. Also provided is a cell transfected with this recombinantexpression system.

[0015] The present invention also provides a polypeptide encoded byPS133. The polypeptide can be produced by recombinant technology,provided in purified form, or produced by synthetic techniques. Thepolypeptide comprises an amino acid sequence which has at least 50%identity with an amino acid sequence selected from the group consistingof SEQUENCE ID NO 24, SEQUENCE ID NO 25, SEQUENCE ID NO 26, and SEQUENCEED NO 27, or at least 80% identity with a fragment of SEQUENCE ID NO 24comprising at least 17 amino acid residues. The polypeptide sharessignificant homology with catalytically functional sites of members ofthe human serine protease family of molecules and is thus consideredherein to be a serine protease.

[0016] Also provided is an antibody which specifically binds to at leastone PS133 epitope. The antibody can be a polyclonal or monoclonalantibody. The epitope is derived from an amino acid sequence selectedfrom the group consisting of SEQUENCE ID NO 24, SEQUENCE ID NO 25,SEQUENCE ID NO 26, SEQUENCE ID NO 27, and fragments thereof. Assay kitsfor determining the presence of PS133 antigen or anti-PS133 antibody ina test sample are also included. In one embodiment, the assay kitscomprise a container containing at least one PS133 polypeptide having atleast 50% identity to an amino acid sequence selected from the groupconsisting of SEQUENCE ID NO 24, SEQUENCE ID NO 25, SEQUENCE ID NO 26,SEQUENCE ID NO 27, and fragments thereof. Further, the test kit cancomprise a container with tools useful for collecting test samples (suchas blood, urine, saliva and stool). Such tools include lancets andabsorbent paper or cloth for collecting and stabilizing blood; swabs forcollecting and stabilizing saliva; and cups for collecting andstabilizing urine or stool samples. Collection materials such as,papers, cloths, swabs, cups and the like, may optionally be treated toavoid denaturation or irreversible adsorption of the sample. Thesecollection materials also may be treated with or contain preservatives,stabilizers or antimicrobial agents to help maintain the integrity ofthe specimens. Also, the polypeptide can be attached to a solid phase.

[0017] Another assay kit for determining the presence of PS133 antigenor anti-PS133 antibody in a test sample comprises a container containingan antibody which specifically binds to a PS133 antigen, wherein thePS133 antigen comprises at least one PS133-encoded epitope. The PS133antigen has at least about 60% sequence similarity to a sequence of aPS133-encoded antigen selected from the group consisting of SEQUENCE IDNO 24, SEQUENCE ID NO 25, SEQUENCE ID NO 26, SEQUENCE ID NO 27, andfragments thereof. These test kits can further comprise containers withtools useful for collecting test samples (such as blood, urine, salivaand stool). Such tools include lancets and absorbent paper or cloth forcollecting and stabilizing blood; swabs for collecting and stabilizingsaliva; cups for collecting and stabilizing urine or stool samples.Collection materials, papers, cloths, swabs, cups and the like, mayoptionally be treated to avoid denaturation or irreversible adsorptionof the sample. These collection materials also may be treated with, orcontain, preservatives, stabilizers or antimicrobial agents to helpmaintain the integrity of the specimens. The antibody can be attached toa solid phase.

[0018] A method for producing a polypeptide which contains at least oneepitope of PS133 is provided, which method comprises incubating hostcells transfected with an expression vector. This vector comprises apolynucleotide sequence encoding a polypeptide, wherein the polypeptidecomprises an amino acid sequence having at least 50% identity to a PS133amino acid sequence selected from the group consisting of SEQUENCE ID NO24, SEQUENCE ID NO 25, SEQUENCE ID NO 26, SEQUENCE ID NO 27, andfragments thereof.

[0019] A method for detecting PS133 antigen in a test sample suspectedof containing PS133 antigen also is provided. The method comprisescontacting the test sample with an antibody or fragment thereof whichspecifically binds to at least one epitope of a PS133 antigen, for atime and under conditions sufficient for the formation ofantibody/antigen complexes; and detecting the presence of such complexescontaining the antibody as an indication of the presence of PS133antigen in the test sample. The antibody can be attached to a solidphase and be either a monoclonal or polyclonal antibody. Furthermore,the antibody specifically binds to at least one PS133 antigen selectedfrom the group consisting of SEQUENCE ID NO 24, SEQUENCE ID NO 25,SEQUENCE ID NO 26, SEQUENCE ID NO 27, and fragments thereof.

[0020] Another method is provided which detects antibodies whichspecifically bind to PS133 antigen in a test sample suspected ofcontaining these antibodies. The method comprises contacting the testsample with a polypeptide which contains at least one PS133 epitope,wherein the PS133 epitope comprises an amino acid sequence having atleast 50% identity with an amino acid sequence encoded by a PS133polynucleotide, or a fragment thereof. Contacting is carried out for atime and under conditions sufficient to allow antigen/antibody complexesto form. The method further entails detecting complexes which containthe polypeptide. The polypeptide can be attached to a solid phase.Further, the polypeptide can be a recombinant protein or a syntheticpeptide having at least 50% identity to an amino acid sequence selectedfrom the group consisting of SEQUENCE ID NO 24, SEQUENCE ID NO 25,SEQUENCE ID NO 26, SEQUENCE ID NO 27, and fragments thereof.

[0021] The present invention provides a cell transfected with a PS133nucleic acid sequence that encodes at least one epitope of a PS133antigen, or fragment thereof. The nucleic acid sequence is selected fromthe group consisting of SEQUENCE ID NOS 1-8, and fragments orcomplements thereof.

[0022] A method for producing antibodies to PS133 antigen also isprovided, which method comprises administering to an individual anisolated immunogenic polypeptide or fragment thereof, wherein theisolated immunogenic polypeptide comprises at least one PS133 epitope inan amount sufficient to produce an immune response. The isolated,immunogenic polypeptide comprises an amino acid sequence selected fromthe group consisting of SEQUENCE ID NO 24, SEQUENCE ID NO 25, SEQUENCEID NO 26, SEQUENCE ID NO 27, and fragments thereof.

[0023] Another method for producing antibodies which specifically bindto PS133 antigen is disclosed, which method comprises administering to amammal a plasmid comprising a nucleic acid sequence which encodes atleast one PS133 epitope derived from an amino acid sequence selectedfrom the group consisting of SEQUENCE ID NO 24, SEQUENCE ID NO 25,SEQUENCE ID NO 26, SEQUENCE ID NO 27, and fragments thereof.

[0024] Also provided is a composition of matter that comprises a PS133polynucleotide of at least about 10-12 nucleotides having at least 50%identity to a polynucleotide selected from the group consisting ofSEQUENCE ID NOS 1-5, SEQUENCE ID NO 8, and fragments or complementsthereof, or at least 50% identity with SEQUENCE ID NO 7. The PS133polynucleotide encodes an amino acid sequence having at least one PS133epitope. Another composition of matter provided by the present inventioncomprises a polypeptide with at least one PS133 epitope of about 8-10amino acids. The polypeptide comprises an amino acid sequence having atleast 50% identity with an amino acid sequence selected from the groupconsisting of SEQUENCE ID NO 24, SEQUENCE ID NO 25, SEQUENCE ID NO 26,and SEQUENCE ID NO 27, or at least 80% identity with a fragment ofSEQUENCE ID NO 24 comprising at least 17 amino acid residues. Alsoprovided is a gene or fragment thereof coding for a PS133 polypeptidewhich has at least 50% identity to SEQUENCE ID NO 24, and a gene or afragment thereof comprising DNA having at least 50% identity to SEQUENCEID NO 7 or SEQUENCE ID NO 8.

[0025] A therapeutic compound (pharmaceutical composition) is providedwhich is useful for treating an individual with a disease or conditionassociated with PS133. The therapeutic compound may be an antagonist, aninhibitor or an agonist of the PS133 polypeptide, gene or mRNA. Thecompound may be a peptide, antibody or nucleic acid directed to acteither synergistically or antagonistically with the PS133 polypeptide,gene or mRNA. The compound may also be mixed with a carrier or diluentto stabilize the compound or to facilitate administration of thecompound to an individual with a disease or condition associated withPS133.

[0026] A method is also provided for treating an individual with adisease or condition associated with PS133. The method comprisesadministering a therapeutic compound to an individual by a suitableroute such as orally or parenterally, e.g. rectal, transdermal, depot,subcutaneous, intravenous, intramuscular or intranasal. The route ofadministration is, preferably, such that the compound will be availablesystemically within the individual.

[0027] A method is also provided of discovering an inhibitor,antagonist, or agonist of PS133. The method comprises screeningpeptides, antibodies or oligonucleotides for the ability to inhibit,eliminate or enhance the proteolytic activity of PS133 polypeptide in anassay utilizing a detectably labeled proteolytic substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIGS. 1A-1B show the nucleotide alignment of clones 828846(SEQUENCE ID NO 1), 2346388 (SEQUENCE ID NO 2), 2531505 (SEQUENCE ID NO3), 1725220 (SEQUENCE ID NO 4), 1856238 (SEQUENCE ID NO 5), g1367041(SEQUENCE ID NO 6), and the consensus sequence (SEQUENCE ID NO 7)derived therefrom.

[0029]FIG. 2 shows the contig map depicting the formation of theconsensus nucleotide sequence (SEQUENCE ID NO 7) from the nucleotidealignment of overlapping clones 828846 (SEQUENCE ID NO 1), 2346388(SEQUENCE ID NO 2), 2531505 (SEQUENCE ID NO 3), 1725220 (SEQUENCE ID NO4), 1856238 (SEQUENCE ID NO 5), and g1367041 (SEQUENCE ID NO 6).

[0030]FIGS. 3A-3D show the PS133 polypeptide, SEQUENCE ID NO 24, alignedwith the amino acid sequence of 47 known human serine proteases orserine protease-like proteins, SEQUENCE ID NOS 28-74. The functionalmotifs present in all catalytically functional proteases are marked withan “*”.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides a gene or a fragment thereof whichcodes for a PS133 polypeptide having at least about 50% identity toSEQUENCE ID NO 24. The present invention further encompasses a PS133gene or a fragment thereof comprising DNA which has at least about 50%identity to SEQUENCE ID NO 7 or SEQUENCE ID NO 8. The present inventionalso provides a novel serine protease. Differentially expressed levelsof serine proteases between normal and diseased prostate tissues can beused as a diagnostic and/or prognostic marker for prostate disease,especially prostate cancer. In addition, serine protease genes, mRNAsand proteins are targets for the design and use of therapeutictreatment.

[0032] The present invention provides methods for assaying a test samplefor products of a prostate tissue gene designated as PS133, whichcomprises making cDNA from mRNA in the test sample, and detecting thecDNA as an indication of the presence of prostate tissue gene PS133. Themethod may include an amplification step, wherein one or more portionsof the mRNA from PS133 corresponding to the gene or fragments thereof,is amplified. Methods also are provided for assaying for the translationproducts of PS133. Test samples which may be assayed by the methodsprovided herein include tissues, cells, body fluids and secretions. Thepresent invention also provides reagents such as oligonucleotide primersand polypeptides which are useful in performing these methods.

[0033] Portions of the nucleic acid sequences disclosed herein areuseful as primers for the reverse transcription of RNA or for theamplification of cDNA; or as probes to determine the presence of certainmRNA sequences in test samples. Also disclosed are nucleic acidsequences which permit the production of encoded polypeptide sequenceswhich are useful as standards or reagents in diagnostic immunoassays, astargets for pharmaceutical screening assays and/or as components or astarget sites for various therapies. Monoclonal and polyclonal antibodiesdirected against at least one epitope contained within these polypeptidesequences are useful as delivery agents for therapeutic agents as wellas for diagnostic tests and for screening for diseases or conditionsassociated with PS133, especially prostate cancer. Isolation ofsequences of other portions of the gene of interest can be accomplishedutilizing probes or PCR primers derived from these nucleic acidsequences. This allows additional probes of the mRNA or cDNA of interestto be established, as well as corresponding encoded polypeptidesequences. These additional molecules are useful in detecting,diagnosing, staging, monitoring, prognosticating, preventing ortreating, or determining the predisposition to, diseases and conditionsof the prostate such as prostate cancer, characterized by PS133, asdisclosed herein.

[0034] Techniques for determining amino acid sequence “similarity” arewell-known in the art. In general, “similarity” means the exact aminoacid to amino acid comparison of two or more polypeptides at theappropriate place, where amino acids are identical or possess similarchemical and/or physical properties such as charge or hydrophobicity. Aso-termed “percent similarity” then can be determined between thecompared polypeptide sequences. Techniques for determining nucleic acidand amino acid sequence identity also are well known in the art andinclude determining the nucleotide sequence of the mRNA for that gene(usually via a cDNA intermediate) and determining the amino acidsequence encoded thereby, and comparing this to a second amino acidsequence. In general, “identity” refers to an exact nucleotide tonucleotide or amino acid to amino acid correspondence of twopolynucleotides or polypeptide sequences, respectively. Two or morepolynucleotide sequences can be compared by determining their “percentidentity.” Two or more amino acid sequences likewise can be compared bydetermining their “percent identity.” The programs available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.), for example, the GAP program, arecapable of calculating both the identity between two polynucleotides andthe identity and similarity between two polypeptide sequences,respectively. Other programs for calculating identity or similaritybetween sequences are known in the art.

[0035] The compositions and methods described herein will enable theidentification of certain markers as indicative of a prostate tissuedisease or condition; the information obtained therefrom will aid in thedetecting, diagnosing, staging, monitoring, prognosticating, preventingor treating, or determining diseases or conditions associated with PS133especially prostate cancer. Test methods include, for example, probeassays which utilize the sequence(s) provided herein and which also mayutilize nucleic acid amplification methods such as the polymerase chainreaction (PCR), the ligase chain reaction (LCR), and hybridization. Inaddition, the nucleotide sequences provided herein contain open readingframes from which an immunogenic epitope may be found. This epitope isbelieved to be unique to the disease state or condition associated withPS133. It also is thought that the polynucleotides or polypeptides andprotein encoded by the PS133 gene are useful as a marker. This marker iseither elevated in disease such as prostate cancer, altered in diseasesuch as prostate cancer, or present as a normal protein but appearing inan inappropriate body compartment. The uniqueness of the epitope may bedetermined by (i) its immunological reactivity and specificity withantibodies directed against proteins and polypeptides encoded by thePS133 gene, and (ii) its nonreactivity with any other tissue markers.Methods for determining immunological reactivity are well-known andinclude but are not limited to, for example, radioimmunoassay (RIA),enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA),fluorescence polarization immunoassay (FPIA), chemiluminescentimmunoassay (CLIA) and others. Several examples of suitable methods aredescribed herein.

[0036] Unless otherwise stated, the following terms shall have thefollowing meanings:

[0037] A polynucleotide “derived from” or “specific for” a designatedsequence refers to a polynucleotide sequence which comprises acontiguous sequence of approximately at least about 6 nucleotides,preferably at least about 8 nucleotides, more preferably at least about10-12 nucleotides, and even more preferably at least about 15-20nucleotides corresponding, i.e., identical or complementary to, a regionof the designated nucleotide sequence. The sequence may be complementaryor identical to a sequence which is unique to a particularpolynucleotide sequence as determined by techniques known in the art.Comparisons to sequences in databanks, for example, can be used as amethod to determine the uniqueness of a designated sequence. Regionsfrom which sequences may be derived, include but are not limited to,regions encoding specific epitopes, as well as non-translated and/ornon-transcribed regions.

[0038] The derived polynucleotide will not necessarily be derivedphysically from the nucleotide sequence of interest under study, but maybe generated in any manner, including, but not limited to, chemicalsynthesis, replication, reverse transcription or transcription, which isbased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived. As such, it mayrepresent either a sense or an antisense orientation of the originalpolynucleotide. In addition, combinations of regions corresponding tothat of the designated sequence may be modified in ways known in the artto be consistent with the intended use.

[0039] A “fragment” of a specified polynucleotide refers to apolynucleotide sequence which comprises a contiguous sequence ofapproximately at least about 6 nucleotides, preferably at least about 8nucleotides, more preferably at least about 10-12 nucleotides, and evenmore preferably at least about 15-20 nucleotides corresponding, i.e.,identical or complementary to, a region of the specified nucleotidesequence.

[0040] The term “primer” denotes a specific oligonucleotide sequencewhich is complementary to a target nucleotide sequence and used tohybridize to the target nucleotide sequence. A primer serves as aninitiation point for nucleotide polymerization catalyzed by either DNApolymerase, RNA polymerase or reverse transcriptase.

[0041] The term “probe” denotes a defined nucleic acid segment (ornucleotide analog segment, e.g., PNA as defined hereinbelow) which canbe used to identify a specific polynucleotide present in samples bearingthe complementary sequence.

[0042] “Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences which areimmunologically identifiable with a polypeptide encoded by the sequence.Thus, a “polypeptide,” “protein,” or “amino acid” sequence has at leastabout 50% identity, preferably about 60% identity, more preferably about75-85% identity, and most preferably about 90-95% or more identity to aPS133 amino acid sequence. Further, the PS133 “polypeptide,” “protein,”or “amino acid” sequence may have at least about 60% similarity,preferably at least about 75% similarity, more preferably about 85%similarity, and most preferably about 95% or more similarity to apolypeptide or amino acid sequence of PS133. This amino acid sequencecan be selected from the group consisting of SEQUENCE ID NO 24, SEQUENCEID NO 25, SEQUENCE ID NO 26, SEQUENCE ID NO 27, and fragments thereof.

[0043] A “recombinant polypeptide,” “recombinant protein,” or “apolypeptide produced by recombinant techniques,” which terms may be usedinterchangeably herein, describes a polypeptide which by virtue of itsorigin or manipulation is not associated with all or a portion of thepolypeptide with which it is associated in nature and/or is linked to apolypeptide other than that to which it is linked in nature. Arecombinant or encoded polypeptide or protein is not necessarilytranslated from a designated nucleic acid sequence. It also may begenerated in any manner, including chemical synthesis or expression of arecombinant expression system.

[0044] The term “synthetic peptide” as used herein means a polymericform of amino acids of any length, which may be chemically synthesizedby methods well-known to the routineer. These synthetic peptides areuseful in various applications.

[0045] The term “polynucleotide” as used herein means a polymeric formof nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes double- and single-stranded DNA,as well as double- and single-stranded RNA. It also includesmodifications, such as methylation or capping and unmodified forms ofthe polynucleotide. The terms “polynucleotide,” “oligomer,”“oligonucleotide,” and “oligo” are used interchangeably herein.

[0046] “A sequence corresponding to a cDNA” means that the sequencecontains a polynucleotide sequence that is identical or complementary toa sequence in the designated DNA. The degree (or “percent”) of identityor complementarity to the cDNA will be approximately 50% or greater,preferably at least about 70% or greater, and more preferably at leastabout 90% or greater. The sequence that corresponds to the identifiedcDNA will be at least about 50 nucleotides in length, preferably atleast about 60 nucleotides in length, and more preferably at least about70 nucleotides in length. The correspondence between the gene or genefragment of interest and the cDNA can be determined by methods known inthe art and include, for example, a direct comparison of the sequencedmaterial with the cDNAs described, or hybridization and digestion withsingle strand nucleases, followed by size determination of the digestedfragments.

[0047] “Purified polynucleotide” refers to a polynucleotide of interestor fragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying-polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

[0048] “Purified polypeptide” or “purified protein” means a polypeptideof interest or fragment thereof which is essentially free of, e.g.,contains less than about 50%, preferably less than about 70%, and morepreferably less than about 90%, cellular components with which thepolypeptide of interest is naturally associated. Methods for purifyingpolypeptides of interest are known in the art.

[0049] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, which is separated from some orall of the coexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

[0050] “Polypeptide” and “protein” are used interchangeably herein andindicate at least one molecular chain of amino acids linked throughcovalent and/or non-covalent bonds. The terms do not refer to a specificlength of the product. Thus peptides, oligopeptides and proteins areincluded within the definition of polypeptide. The terms includepost-translational modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Inaddition, protein fragments, analogs, mutated or variant proteins,fusion proteins and the like are included within the meaning ofpolypeptide.

[0051] A “fragment” of a specified polypeptide refers to an amino acidsequence which comprises at least about 3-5 amino acids, more preferablyat least about 8-10 amino acids, and even more preferably at least about17-20 amino acids derived from the specified polypeptide.

[0052] “Recombinant host cells,” “host cells,” “cells,” “cell lines,”“cell cultures,” and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be, or have been, used as recipients for recombinant vector orother transferred DNA, and include the original progeny of the originalcell which has been transfected.

[0053] As used herein “replicon” means any genetic element, such as aplasmid, a chromosome or a virus, that behaves as an autonomous unit ofpolynucleotide replication within a cell.

[0054] A “vector” is a replicon in which another polynucleotide segmentis attached, such as to bring about the replication and/or expression ofthe attached segment.

[0055] The term “control sequence” refers to a polynucleotide sequencewhich is necessary to effect the expression of a coding sequence towhich it is ligated. The nature of such control sequences differsdepending upon the host organism. In prokaryotes, such control sequencesgenerally include a promoter, a ribosomal binding site and terminators;in eukaryotes, such control sequences generally include promoters,terminators and, in some instances, enhancers. The term “controlsequence” thus is intended to include at a minimum all components whosepresence is necessary for expression, and also may include additionalcomponents whose presence is advantageous, for example, leadersequences.

[0056] “Operably linked” refers to a situation wherein the componentsdescribed are in a relationship permitting them to function in theirintended manner. Thus, for example, a control sequence “operably linked”to a coding sequence is ligated in such a manner that expression of thecoding sequence is achieved under conditions compatible with the controlsequence.

[0057] The term “open reading frame” or “ORE” refers to a region of apolynucleotide sequence which encodes a polypeptide. This region mayrepresent a portion of a coding sequence or a total coding sequence.

[0058] A “coding sequence” is a polynucleotide sequence which istranscribed into mRNA and translated into a polypeptide when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a translation start codon at the5′-terminus and a translation stop codon at the 3′-terminus. A codingsequence can include, but is not limited to, mRNA, cDNA and recombinantpolynucleotide sequences.

[0059] The term “immunologically identifiable with/as” refers to thepresence of epitope(s) and polypeptide(s) which also are present in andare unique to the designated polypeptide(s). Immunological identity maybe determined by antibody binding and/or competition in binding. Thesetechniques are known to the routineer and also are described herein. Theuniqueness of an epitope also can be determined by computer searches ofknown data banks, such as GenBank, for the polynucleotide sequence whichencodes the epitope and by amino acid sequence comparisons with otherknown proteins.

[0060] As used herein, “epitope” means an antigenic determinant of apolypeptide or protein. Conceivably, an epitope can comprise three aminoacids in a spatial conformation which is unique to the epitope.Generally, an epitope consists of at least five such amino acids andmore usually, it consists of at least eight to ten amino acids. Methodsof examining spatial conformation are known in the art and include, forexample, x-ray crystallography and two-dimensional nuclear magneticresonance.

[0061] A “conformational epitope” is an epitope that is comprised of aspecific juxtaposition of amino acids in an immunologically recognizablestructure, such amino acids being present on the same polypeptide in acontiguous or non-contiguous order or present on different polypeptides.

[0062] A polypeptide is “immunologically reactive” with an antibody whenit binds to an antibody due to antibody recognition of a specificepitope contained within the polypeptide. Immunological reactivity maybe determined by antibody binding, more particularly, by the kinetics ofantibody binding, and/or by competition in binding using ascompetitor(s) a known polypeptide(s) containing an epitope against whichthe antibody is directed. The methods for determining whether apolypeptide is immunologically reactive with an antibody are known inthe art.

[0063] As used herein, the term “immunogenic polypeptide containing anepitope of interest” means naturally occurring polypeptides of interestor fragments thereof, as well as polypeptides prepared by other means,for example, by chemical synthesis or the expression of the polypeptidein a recombinant organism.

[0064] The term “transfection” refers to the introduction of anexogenous polynucleotide into a prokaryotic or eucaryotic host cell,irrespective of the method used for the introduction. The term“transfection” refers to both stable and transient introduction of thepolynucleotide, and encompasses direct uptake of polynucleotides,transformation, transduction, and f-mating. Once introduced into thehost cell, the exogenous polynucleotide may be maintained as anon-integrated replicon, for example, a plasmid, or alternatively, maybe integrated into the host genome.

[0065] “Treatment” refers to prophylaxis and/or therapy.

[0066] The term “individual” as used herein refers to vertebrates,particularly members of the mammalian species and includes, but is notlimited to, domestic animals, sports animals, primates and humans; moreparticularly, the term refers to humans.

[0067] The term “sense strand” or “plus strand” (or “+”) as used hereindenotes a nucleic acid that contains the sequence that encodes thepolypeptide. The term “antisense strand” or “minus strand” (or “−”)denotes a nucleic acid that contains a sequence that is complementary tothat of the “plus” strand.

[0068] The term “test sample” refers to a component of an individual'sbody which is the source of the analyte (such as, antibodies of interestor antigens of interest). These components are well known in the art. Atest sample is typically anything suspected of containing a targetsequence. Test samples can be prepared using methodologies well known inthe art such as by obtaining a specimen from an individual and, ifnecessary, disrupting any cells contained thereby to release targetnucleic acids. These test samples include biological samples which canbe tested by the methods of the present invention described herein andinclude human and animal body fluids such as whole blood, serum, plasma,cerebrospinal fluid, sputum, bronchial washing, bronchial aspirates,urine, lymph fluids and various external secretions of the respiratory,intestinal and genitourinary tracts, tears, saliva, milk, white bloodcells, myelomas and the like; biological fluids such as cell culturesupernatants; tissue specimens which may be fixed; and cell specimenswhich may be fixed.

[0069] “Purified product” refers to a preparation of the product whichhas been isolated from the cellular constituents with which the productis normally associated and from other types of cells which may bepresent in the sample of interest.

[0070] “PNA” denotes a “peptide nucleic acid analog” which may beutilized in a procedure such as an assay described herein to determinethe presence of a target. “MA” denotes a “morpholino analog” which maybe utilized in a procedure such as an assay described herein todetermine the presence of a target. See, for example, U.S. Pat. No.5,378,841, which is incorporated herein by reference. PNAs are neutrallycharged moieties which can be directed against RNA targets or DNA. PNAprobes used in assays in place of, for example, the DNA probes of thepresent invention, offer advantages not achievable when DNA probes areused. These advantages include manufacturability, large scale labeling,reproducibility, stability, insensitivity to changes in ionic strengthand resistance to enzymatic degradation which is present in methodsutilizing DNA or RNA. These PNAs can be labeled with (“attached to”)such signal generating compounds as fluorescein, radionucleotides,chemiluminescent compounds and the like. PNAs or other nucleic acidanalogs such as MAs thus can be used in assay methods in place of DNA orRNA. Although assays are described herein utilizing DNA probes, it iswithin the scope of the routineer that PNAs or MAs can be substitutedfor RNA or DNA with appropriate changes if and as needed in assayreagents.

[0071] “Analyte,” as used herein, is the substance to be detected whichmay be present in the test sample. The analyte can be any substance forwhich there exists a naturally occurring specific binding member (suchas, an antibody), or for which a specific binding member can beprepared. Thus, an analyte is a substance that can bind to one or morespecific binding members in an assay. “Analyte” also includes anyantigenic substances, haptens, antibodies and combinations thereof. As amember of a specific binding pair, the analyte can be detected by meansof naturally occurring specific binding partners (pairs) such as the useof intrinsic factor protein as a member of a specific binding pair forthe determination of Vitamin B12, the use of folate-binding protein todetermine folic acid, or the use of a lectin as a member of a specificbinding pair for the determination of a carbohydrate. The analyte caninclude a protein, a polypeptide, an amino acid, a nucleotide target andthe like.

[0072] “Diseases of the prostate” or “prostate disease,” or “conditionof the prostate,” as used herein, refer to any disease or condition ofthe prostate including, but not limited to, benign prostatic hyperplasia(BPH), prostatitis, prostatic intraepithelial neoplasia (PIN) andcancer.

[0073] “Prostate cancer,” as used herein, refers to any malignantdisease of the prostate including, but not limited to, adenocarcinoma,small cell undifferentiated carcinoma and mucinous (colloid) cancer.

[0074] An “Expressed Sequence Tag” or “EST” refers to the partialsequence of a cDNA insert which has been made by reverse transcriptionof mRNA extracted from a tissue followed by insertion into a vector.

[0075] A “transcript image” refers to a table or list giving thequantitative distribution of ESTs in a library and represents the genesactive in the tissue from which the library was made.

[0076] The present invention provides assays which utilize specificbinding members. A “specific binding member,” as used herein, is amember of a specific binding pair. That is, two different moleculeswhere one of the molecules, through chemical or physical means,specifically binds to the second molecule. Therefore, in addition toantigen and antibody specific binding pairs of common immunoassays,other specific binding pairs can include biotin and avidin,carbohydrates and lectins, complementary nucleotide sequences, effectorand receptor molecules, cofactors and enzymes, enzyme inhibitors, andenzymes and the like. Furthermore, specific binding pairs can includemembers that are analogs of the original specific binding members, forexample, an analyte-analog. Immunoreactive specific binding membersinclude antigens, antigen fragments, antibodies and antibody fragments,both monoclonal and polyclonal and complexes thereof, including thoseformed by recombinant DNA molecules.

[0077] The term “hapten,” as used herein, refers to a partial antigen ornon-protein binding member which is capable of binding to an antibody,but which is not capable of eliciting antibody formation unless coupledto a carrier protein.

[0078] A “capture reagent,” as used herein, refers to an unlabeledspecific binding member which is specific either for the analyte as in asandwich assay, for the indicator reagent or analyte as in a competitiveassay, or for an ancillary specific binding member, which itself isspecific for the analyte, as in an indirect assay. The capture reagentcan be directly or indirectly bound to a solid phase material before theperformance of the assay or during the performance of the assay, therebyenabling the separation of immobilized complexes from the test sample.

[0079] The “indicator reagent” comprises a “signal-generating compound”(“label”) which is capable of generating and generates a measurablesignal detectable by external means, conjugated (“attached”) to aspecific binding member. In addition to being an antibody member of aspecific binding pair, the indicator reagent also can be a member of anyspecific binding pair, including either hapten-anti-hapten systems suchas biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin,a complementary nucleotide sequence, an effector or a receptor molecule,an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme andthe like. An immunoreactive specific binding member can be an antibody,an antigen, or an antibody/antigen complex that is capable of bindingeither to the polypeptide of interest as in a sandwich assay, to thecapture reagent as in a competitive assay, or to the ancillary specificbinding member as in an indirect assay. When describing probes and probeassays, the term “reporter molecule” may be used. A reporter moleculecomprises a signal generating compound as described hereinaboveconjugated to a specific binding member of a specific binding pair, suchas carbazole or adamantane.

[0080] The various “signal-generating compounds” (labels) contemplatedinclude chromagens, catalysts such as enzymes, luminescent compoundssuch as fluorescein and rhodamine, chemiluminescent compounds such asdioxetanes, acridiniums, phenanthridiniums and luminol, radioactiveelements and direct visual labels. Examples of enzymes include alkalinephosphatase, horseradish peroxidase, beta-galactosidase and the like.The selection of a particular label is not critical, but it must becapable of producing a signal either by itself or in conjunction withone or more additional substances.

[0081] “Solid phases” (“solid supports”) are known to those in the artand include the walls of wells of a reaction tray, test tubes,polystyrene beads, magnetic or non-magnetic beads, nitrocellulosestrips, membranes, microparticles such as latex particles, sheep (orother animal) red blood cells and Duracytes® (red blood cells “fixed” bypyruvic aldehyde and formaldehyde, available from Abbott Laboratories,Abbott Park, Ill.) and others. The “solid phase” is not critical and canbe selected by one skilled in the art. Thus, latex particles,microparticles, magnetic or non-magnetic beads, membranes, plastictubes, walls of microtiter wells, glass or silicon chips, sheep (orother suitable animal's) red blood cells and Duracytes® are all suitableexamples. Suitable methods for immobilizing peptides on solid phasesinclude ionic, hydrophobic, covalent interactions and the like. A “solidphase,” as used herein, refers to any material which is insoluble, orcan be made insoluble by a subsequent reaction. The solid phase can bechosen for its intrinsic ability to attract and immobilize the capturereagent. Alternatively, the solid phase can retain an additionalreceptor which has the ability to attract and immobilize the capturereagent. The additional receptor can include a charged substance that isoppositely charged with respect to the capture reagent itself or to acharged substance conjugated to the capture reagent. As yet anotheralternative, the receptor molecule can be any specific binding memberwhich is immobilized upon (attached to) the solid phase and which hasthe ability to immobilize the capture reagent through a specific bindingreaction. The receptor molecule enables the indirect binding of thecapture reagent to a solid phase material before the performance of theassay or during the performance of the assay. The solid phase thus canbe a plastic, derivatized plastic, magnetic or non-magnetic metal, glassor silicon surface of a test tube, microtiter well, sheet, bead,microparticle, chip, sheep (or other suitable animal's) red blood cells,Duracytes® and other configurations known to those of ordinary skill inthe art.

[0082] It is contemplated and within the scope of the present inventionthat the solid phase also can comprise any suitable porous material withsufficient porosity to allow access by detection antibodies and asuitable surface affinity to bind antigens. Microporous structuresgenerally are preferred, but materials with a gel structure in thehydrated state may be used as well. Such useful solid supports include,but are not limited to, nitrocellulose and nylon. It is contemplatedthat such porous solid supports described herein preferably are in theform of sheets of thickness from about 0.01 to 0.5 mm, preferably about0.1 mm. The pore size may vary within wide limits and preferably is fromabout 0.025 to 15 microns, especially from about 0.15 to 15 microns. Thesurface of such supports may be activated by chemical processes whichcause covalent linkage of the antigen or antibody to the support. Theirreversible binding of the antigen or antibody is obtained, however, ingeneral, by adsorption on the porous material by poorly understoodhydrophobic forces. Other suitable solid supports are known in the art.

[0083] The terms “therapeutically effective dose” and “efficacious dose”of a drug, agent, or compound, are used interchangeably herein to definean amount of a particular drug, agent or compound to be administered toa patient suffering from prostate disease, especially prostate cancer,within the context of a suitable therapeutic regimen that is sufficientto cure, partially arrest, or detectably slow the progression of thedisease and its complications.

[0084] A “pharmaceutical carrier” or “pharmaceutical diluent” arepharmaceutically-acceptable, non-toxic vehicles commonly used toformulate pharmaceutical compositions for animal or humanadministration.

[0085] A “serine protease,” as described herein, is any peptidecontaining three functional motifs, in the following order and spacing:a His motif (approximately 10-40 amino acid residues from the aminoterminus), an Asp motif (approximately 20-75 residues further in fromthe His motif toward the carboxy terminus), and a Ser motif(approximately 60-140 residues further in from the Asp motif toward thecarboxy terminus), each motif being named after the catalytic residuewithin each active site of the motif.

[0086] Reagents.

[0087] The present invention provides reagents such as polynucleotidesequences derived from a prostate tissue of interest and designated asPS133, polypeptides encoded thereby and antibodies specific for thesepolypeptides. The present invention also provides reagents such asoligonucleotide fragments derived from the disclosed polynucleotides andnucleic acid sequences complementary to these polynucleotides. Thepolynucleotides, polypeptides, or antibodies of the present inventionmay be used to provide information leading to the detecting, diagnosing,staging, monitoring, prognosticating, preventing or treating of, ordetermining the predisposition to, diseases and conditions of theprostate such as cancer. The sequences disclosed herein represent uniquepolynucleotides which can be used in assays or for producing a specificprofile of gene transcription activity. Such assays are disclosed inEuropean Patent Number 0373203B1 and International Publication No. WO95/11995, which are hereby incorporated by reference.

[0088] Selected PS133-derived polynucleotides can be used in the methodsdescribed herein for the detection of normal or altered gene expression.Such methods may employ PS133 polynucleotides or oligonucleotides,fragments or derivatives thereof, or nucleic acid sequencescomplementary thereto.

[0089] The polynucleotides disclosed herein, their complementarysequences, or fragments of either, can be used in assays to detect,amplify or quantify genes, nucleic acids, cDNAs or mRNAs relating toprostate tissue disease and conditions associated therewith. They alsocan be used to identify an entire or partial coding region of a PS133polypeptide. They further can be provided in individual containers inthe form of a kit for assays, or provided as individual compositions. Ifprovided in a kit for assays, other suitable reagents such as buffers,conjugates and the like may be included.

[0090] The polynucleotide may be in the form of RNA or DNA.Polynucleotides in the form of DNA, cDNA, genomic DNA, nucleic acidanalogs and synthetic DNA are within the scope of the present invention.The DNA may be double-stranded or single-stranded, and if singlestranded, may be the coding (sense) strand or non-coding (anti-sense)strand. The coding sequence which encodes the polypeptide may beidentical to the coding sequence provided herein or may be a differentcoding sequence which coding sequence, as a result of the redundancy ordegeneracy of the genetic code, encodes the same polypeptide as the DNAprovided herein.

[0091] This polynucleotide may include only the coding sequence for thepolypeptide, or the coding sequence for the polypeptide and anadditional coding sequence such as a leader or secretory sequence or aproprotein sequence, or the coding sequence for the polypeptide (andoptionally an additional coding sequence) and non-coding sequence, suchas a non-coding sequence 5′ and/or 3′ of the coding sequence for thepolypeptide.

[0092] In addition, the invention includes variant polynucleotidescontaining modifications such as polynucleotide deletions, substitutionsor additions; and any polypeptide modification resulting from thevariant polynucleotide sequence. A polynucleotide of the presentinvention also may have a coding sequence which is a naturally occurringallelic variant of the coding sequence provided herein.

[0093] In addition, the coding sequence for the polypeptide may be fusedin the same reading frame to a polynucleotide sequence which aids inexpression and secretion of a polypeptide from a host cell, for example,a leader sequence which functions as a secretory sequence forcontrolling transport of a polypeptide from the cell. The polypeptidehaving a leader sequence is a preprotein and may have the leadersequence cleaved by the host cell to form the polypeptide. Thepolynucleotides may also encode for a proprotein which is the proteinplus additional 5′ amino acid residues. A protein having a prosequenceis a proprotein and may, in some cases, be an inactive form of theprotein. Once the prosequence is cleaved, an active protein remains.Thus, the polynucleotide of the present invention may encode for aprotein, or for a protein having a prosequence, or for a protein havingboth a presequence (leader sequence) and a prosequence.

[0094] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the polypeptide fused to the marker in thecase of a bacterial host, or, for example, the marker sequence may be ahemagglutinin (HA) tag when a mammalian host, e.g. a COS-7 cell line, isused. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein. See, for example, I. Wilson, et al., Cell 37:767(1984).

[0095] It is contemplated that polynucleotides will be considered tohybridize to the sequences provided herein if there is at least 50%,preferably at least 70%, and more preferably at least 90% identitybetween the polynucleotide and the sequence.

[0096] The present invention also provides an antibody produced by usinga purified PS133 polypeptide of which at least a portion of thepolypeptide is encoded by a PS133 polynucleotide selected from thepolynucleotides provided herein. These antibodies may be used in themethods provided herein for the detection of PS133 antigen in testsamples. The presence of PS133 antigen in the test samples is indicativeof the presence of a prostate disease or condition. The antibody alsomay be used for therapeutic purposes, for example, in neutralizing theactivity of PS133 polypeptide in conditions associated with altered orabnormal expression.

[0097] The present invention further relates to a PS133 polypeptidewhich has the deduced amino acid sequence as provided herein, as well asfragments, analogs and derivatives of such polypeptide. The polypeptideof the present invention may be a recombinant polypeptide, a naturalpurified polypeptide or a synthetic polypeptide. The fragment,derivative or analog of the PS133 polypeptide may be one in which one ormore of the amino acid residues is substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code; or it may be one in which one or more ofthe amino acid residues includes a substituent group; or it may be onein which the polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol); or it may be one in which the additional aminoacids are fused to the polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of thepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are within the scope of the present invention. The polypeptidesand polynucleotides of the present invention are provided preferably inan isolated form and preferably purified.

[0098] Thus, a polypeptide of the present invention may have an aminoacid sequence that is identical to that of the naturally occurringpolypeptide or that is different by minor variations due to one or moreamino acid substitutions. The variation may be a “conservative change”typically in the range of about 1 to 5 amino acids, wherein thesubstituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine or threonine with serine.In contrast, variations may include nonconservative changes, e.g.,replacement of a glycine with a tryptophan. Similar minor variations mayalso include amino acid deletions or insertions, or both. Guidance indetermining which and how many amino acid residues may be substituted,inserted or deleted without changing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software (DNASTAR Inc., Madison Wis.).

[0099] Probes constructed according to the polynucleotide sequences ofthe present invention can be used in various assay methods to providevarious types of analysis. For example, such probes can be used influorescent in situ hybridization (FISH) technology to performchromosomal analysis, and used to identify cancer-specific structuralalterations in the chromosomes, such as deletions or translocations thatare visible from chromosome spreads or detectable using PCR-generatedand/or allele specific oligonucleotides probes, allele specificamplification or by direct sequencing. Probes also can be labeled withradioisotopes, directly- or indirectly-detectable haptens, orfluorescent molecules, and utilized for in situ hybridization studies toevaluate the mRNA expression of the gene comprising the polynucleotidein tissue specimens or cells.

[0100] This invention also provides teachings as to the production ofthe polynucleotides and polypeptides provided herein.

[0101] Probe Assays

[0102] The sequences provided herein may be used to produce probes whichcan be used in assays for the detection of nucleic acids in testsamples. The probes may be designed from conserved nucleotide regions ofthe polynucleotides of interest or from non-conserved nucleotide regionsof the polynucleotide of interest. The design of such probes foroptimization in assays is within the skill of the routineer. Generally,nucleic acid probes are developed from non-conserved or unique regionswhen maximum specificity is desired, and nucleic acid probes aredeveloped from conserved regions when assaying for nucleotide regionsthat are closely related to, for example, different members of amulti-gene family or in related species like mouse and man.

[0103] The polymerase chain reaction (PCR) is a technique for amplifyinga desired nucleic acid sequence (target) contained in a nucleic acid ormixture thereof. In PCR, a pair of primers are employed in excess tohybridize to the complementary strands of the target nucleic acid. Theprimers are each extended by a polymerase using the target nucleic acidas a template. The extension products become target sequencesthemselves, following dissociation from the original target strand. Newprimers then are hybridized and extended by a polymerase, and the cycleis repeated to geometrically increase the number of target sequencemolecules. PCR is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202,which are incorporated herein by reference.

[0104] The Ligase Chain Reaction (LCR) is an alternate method fornucleic acid amplification. In LCR, probe pairs are used which includetwo primary (first and second) and two secondary (third and fourth)probes, all of which are employed in molar excess to target. The firstprobe hybridizes to a first segment of the target strand, and the secondprobe hybridizes to a second segment of the target strand, the first andsecond segments being contiguous so that the primary probes abut oneanother in 5′ phosphate-3′ hydroxyl relationship, and so that a ligasecan covalently fuse or ligate the two probes into a fused product. Inaddition, a third (secondary) probe can hybridize to a portion of thefirst probe and a fourth (secondary) probe can hybridize to a portion ofthe second probe in a similar abutting fashion. Of course, if the targetis initially double stranded, the secondary probes also will hybridizeto the target complement in the first instance. Once the ligated strandof primary probes is separated from the target strand, it will hybridizewith the third and fourth probes which can be ligated to form acomplementary, secondary ligated product. It is important to realizethat the ligated products are functionally equivalent to either thetarget or its complement. By repeated cycles of hybridization andligation, amplification of the target sequence is achieved. Thistechnique is described more completely in EP-A-320 308 to K. Backmanpublished Jun. 16, 1989 and EP-A-439 182 to K. Backman et al., publishedJul. 31, 1991, both of which are incorporated herein by reference.

[0105] For amplification of mRNAs, it is within the scope of the presentinvention to reverse transcribe mRNA into cDNA followed by polymerasechain reaction (RT-PCR); or, to use a single enzyme for both steps asdescribed in U.S. Pat. No. 5,322,770, which is incorporated herein byreference; or reverse transcribe mRNA into cDNA followed by asymmetricgap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall etal., PCR Methods and Applications 4: 80-84 (1994), which also isincorporated herein by reference.

[0106] Other known amplification methods which can be utilized hereininclude but are not limited to the so-called “NASBA” or “3SR” techniquedescribed by J. C. Guatelli et al., PNAS USA 87:1874-1878 (1990) andalso described by J. Compton, Nature 350 (No. 6313):91-92 (1991); Q-betaamplification as described in published European Patent Application(EPA) No. 4544610; strand displacement amplification (as described in G.T. Walker et al., Clin. Chem. 42:9-13 [1996]) and European PatentApplication No. 684315; and target mediated amplification, as describedin International Publication No. WO 93/22461.

[0107] Detection of PS133 may be accomplished using any suitabledetection method, including those detection methods which are currentlywell known in the art, as well as detection strategies which may evolvelater. Examples of the foregoing presently known detection methods arehereby incorporated herein by reference. See, for example, Caskey etal., U.S. Pat. No. 5,582,989, Gelfand et al., U.S. Pat. No. 5,210,015.Examples of such detection methods include target amplification methodsas well as signal amplification technologies. An example of presentlyknown detection methods would include the nucleic acid amplificationtechnologies referred to as PCR, LCR, NASBA, SDA, RCR and TMA. See, forexample, Caskey et al., U.S. Pat. No. 5,582,989, Gelfand et al., U.S.Pat. No. 5,210,015. All of the foregoing are hereby incorporated byreference. Detection may also be accomplished using signal amplificationsuch as that disclosed in Snitman et al., U.S. Pat. No. 5,273,882. Whilethe amplification of target or signal is preferred at present, it iscontemplated and within the scope of the present invention thatultrasensitive detection methods which do not require amplification canbe utilized herein.

[0108] Detection, both amplified and non-amplified, may be (combined)carried out using a variety of heterogeneous and homogeneous detectionformats. Examples of heterogeneous detection formats are disclosed inSnitman et al., U.S. Pat. No. 5,273,882, Albarella et al. inEP-84114441.9, Urdea et al., U.S. Pat. No. 5,124,246, Ullman et al. U.S.Pat. No. 5,185,243 and Kourilsky et al., U.S. Pat. No. 4,581,333. All ofthe foregoing are hereby incorporated by reference. Examples ofhomogeneous detection formats are disclosed in, Caskey et al., U.S. Pat.No. 5,582,989, Gelfand et al., U.S. Pat. No. 5,210,015, which areincorporated herein by reference. Also contemplated and within the scopeof the present invention is the use of multiple probes in thehybridization assay, which use improves sensitivity and amplification ofthe PS133 signal. See, for example, Caskey et al., U.S. Pat. No.5,582,989, Gelfand et al., U.S. Pat. No. 5,210,015, which areincorporated herein by reference.

[0109] In one embodiment, the present invention generally comprises thesteps of contacting a test sample suspected of containing a targetpolynucleotide sequence with amplification reaction reagents comprisingan amplification primer, and a detection probe that can hybridize withan internal region of the amplicon sequences. Probes and primersemployed according to the method provided herein are labeled withcapture and detection labels, wherein probes are labeled with one typeof label and primers are labeled with another type of label.Additionally, the primers and probes are selected such that the probesequence has a lower melt temperature than the primer sequences. Theamplification reagents, detection reagents and test sample are placedunder amplification conditions whereby, in the presence of targetsequence, copies of the target sequence (an amplicon) are produced. Inthe usual case, the amplicon is double stranded because primers areprovided to amplify a target sequence and its complementary strand. Thedouble stranded amplicon then is thermally denatured to produce singlestranded amplicon members. Upon formation of the single strandedamplicon members, the mixture is cooled to allow the formation ofcomplexes between the probes and single stranded amplicon members.

[0110] As the single stranded amplicon sequences and probe sequences arecooled, the probe sequences preferentially bind the single strandedamplicon members. This finding is counterintuitive given that the probesequences generally are selected to be shorter than the primer sequencesand therefore have a lower melt temperature than the primers.Accordingly, the melt temperature of the amplicon produced by theprimers should also have a higher melt temperature than the probes.Thus, as the mixture cools, the re-formation of the double strandedamplicon would be expected. As previously stated, however, this is notthe case. The probes are found to preferentially bind the singlestranded amplicon members. Moreover, this preference of probe/singlestranded amplicon binding exists even when the primer sequences areadded in excess of the probes.

[0111] After the probe/single stranded amplicon member hybrids areformed, they are detected. Standard heterogeneous assay formats aresuitable for detecting the hybrids using the detection labels andcapture labels present on the primers and probes. The hybrids can bebound to a solid phase reagent by virtue of the capture label anddetected by virtue of the detection label. In cases where the detectionlabel is directly detectable, the presence of the hybrids on the solidphase can be detected by causing the label to produce a detectablesignal, if necessary, and detecting the signal. In cases where the labelis not directly detectable, the captured hybrids can be contacted with aconjugate, which generally comprises a binding member attached to adirectly detectable label. The conjugate becomes bound to the complexesand the conjugate's presence on the complexes can be detected with thedirectly detectable label. Thus, the presence of the hybrids on thesolid phase reagent can be determined. Those skilled in the art willrecognize that wash steps may be employed to wash away unhybridizedamplicon or probe as well as unbound conjugate.

[0112] Although the target sequence is described as single stranded, italso is contemplated to include the case where the target sequence isactually double stranded but is merely separated from its complementprior to hybridization with the amplification primer sequences. In thecase where PCR is employed in this method, the ends of the targetsequences are usually known. In cases where LCR or a modificationthereof is employed in the preferred method, the entire target sequenceis usually known. Typically, the target sequence is a nucleic acidsequence such as, for example, RNA or DNA.

[0113] The method provided herein can be used in well-knownamplification reactions that include thermal cycle reaction mixtures,particularly in PCR and gap LCR (GLCR). Amplification reactionstypically employ primers to repeatedly generate copies of a targetnucleic acid sequence, which target sequence is usually a small regionof a much larger nucleic acid sequence. Primers are themselves nucleicacid sequences that are complementary to regions of a target sequence.Under amplification conditions, these primers hybridize or bind to thecomplementary regions of the target sequence. Copies of the targetsequence typically are generated by the process of primer extensionand/or ligation which utilizes enzymes with polymerase or ligaseactivity, separately or in combination, to add nucleotides to thehybridized primers and/or ligate adjacent probe pairs. The nucleotidesthat are added to the primers or probes, as monomers or preformedoligomers, are also complementary to the target sequence. Once theprimers or probes have been sufficiently extended and/or ligated, theyare separated from the target sequence, for example, by heating thereaction mixture to a “melt temperature” which is one in whichcomplementary nucleic acid strands dissociate. Thus, a sequencecomplementary to the target sequence is formed.

[0114] A new amplification cycle then can take place to further amplifythe number of target sequences by separating any double strandedsequences, allowing primers or probes to hybridize to their respectivetargets, extending and/or ligating the hybridized primers or probes andre-separating. The complementary sequences that are generated byamplification cycles can serve as templates for primer extension orfilling the gap of two probes to further amplify the number of targetsequences. Typically, a reaction mixture is cycled between 20 and 100times, more typically, a reaction mixture is cycled between 25 and 50times. The numbers of cycles can be determined by the routineer. In thismanner, multiple copies of the target sequence and its complementarysequence are produced. Thus, primers initiate amplification of thetarget sequence when it is present under amplification conditions.

[0115] Generally, two primers which are complementary to a portion of atarget strand and its complement are employed in PCR. For LCR, fourprobes, two of which are complementary to a target sequence and two ofwhich are similarly complementary to the target's complement, aregenerally employed. In addition to the primer sets and enzymespreviously mentioned, a nucleic acid amplification reaction mixture mayalso comprise other reagents which are well known and include but arenot limited to: enzyme cofactors such as manganese; magnesium; salts;nicotinamide adenine dinucleotide (NAD); and deoxynucleotidetriphosphates (dNTPs) such as, for example, deoxyadenine triphosphate,deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythyminetriphosphate.

[0116] While the amplification primers initiate amplification of thetarget sequence, the detection (or hybridization) probe is not involvedin amplification. Detection probes are generally nucleic acid sequencesor uncharged nucleic acid analogs such as, for example, peptide nucleicacids which are disclosed in International Publication No. WO 92/20702;morpholino analogs which are described in U.S. Pat. Nos 5,185,444,5,034,506 and 5,142,047; and the like. Depending upon the type of labelcarried by the probe, the probe is employed to capture or detect theamplicon generated by the amplification reaction. The probe is notinvolved in amplification of the target sequence and therefore may haveto be rendered “non-extendible” in that additional dNTPs cannot be addedto the probe. In and of themselves, analogs usually are non-extendibleand nucleic acid probes can be rendered non-extendible by modifying the3′ end of the probe such that the hydroxyl group is no longer capable ofparticipating in elongation. For example, the 3′ end of the probe can befunctionalized with the capture or detection label to thereby consume orotherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl groupsimply can be cleaved, replaced or modified. U.S. patent applicationSer. No. 07/049,061 filed Apr. 19, 1993 and incorporated herein byreference describes modifications which can be used to render a probenon-extendible.

[0117] The ratio of primers to probes is not important. Thus, either theprobes or primers can be added to the reaction mixture in excess wherebythe concentration of one would be greater than the concentration of theother. Alternatively, primers and probes can be employed in equivalentconcentrations. Preferably, however, the primers are added to thereaction mixture in excess of the probes. Thus, primer to probe ratiosof, for example, 5:1 and 20:1, are preferred.

[0118] While the length of the primers and probes can vary, the probesequences are selected such that they have a lower melt temperature thanthe primer sequences. Hence, the primer sequences are generally longerthan the probe sequences. Typically, the primer sequences are in therange of between 20 and 50 nucleotides long, more typically in the rangeof between 20 and 30 nucleotides long. The typical probe is in the rangeof between 10 and 25 nucleotides long.

[0119] Various methods for synthesizing primers and probes are wellknown in the art. Similarly, methods for attaching labels to primers orprobes are also well known in the art. For example, it is a matter ofroutine to synthesize desired nucleic acid primers or probes usingconventional nucleotide phosphoramidite chemistry and instrumentsavailable from Applied Biosystems, Inc., (Foster City, Calif.), DuPont(Wilmington, Del.), or Milligen (Bedford Mass.). Many methods have beendescribed for labeling oligonucleotides such as the primers or probes ofthe present invention. Enzo Biochemical (New York, N.Y.) and Clontech(Palo Alto, Calif.) both have described and commercialized probelabeling techniques. For example, a primary amine can be attached to a3′ oligo terminus using 3′-Amine-ON CPG™ (Clontech, Palo Alto, Calif.).Similarly, a primary amine can be attached to a 5′ oligo terminus usingAminomodifier II® (Clontech). The amines can be reacted to varioushaptens using conventional activation and linking chemistries. Inaddition, copending applications U.S. Ser. Nos. 625,566, filed Dec. 11,1990 and 630,908, filed Dec. 20, 1990, which are each incorporatedherein by reference, teach methods for labeling probes at their 5′ and3′ termini, respectively. International Publication Nos WO 92/10505,published 25 Jun. 1992, and WO 92/11388, published 9 Jul. 1992, teachmethods for labeling probes at their 5′ and 3′ ends, respectively.According to one known method for labeling an oligonucleotide, alabel-phosphoramidite reagent is prepared and used to add the label tothe oligonucleotide during its synthesis. See, for example, N. T. Thuonget al., Tet. Letters 29(46):5905-5908 (1988); or J. S. Cohen et al.,published U.S. patent application Ser. No. 07/246,688 (NTIS ORDER No.PAT-APPL-7-246,688) (1989). Preferably, probes are labeled at their 3′and 5′ ends.

[0120] A capture label is attached to the primers or probes and can be aspecific binding member which forms a binding pair with the solid phasereagent's specific binding member. It will be understood that the primeror probe itself may serve as the capture label. For example, in the casewhere a solid phase reagent's binding member is a nucleic acid sequence,it may be selected such that it binds a complementary portion of theprimer or probe to thereby immobilize the primer or probe to the solidphase. In cases where the probe itself serves as the binding member,those skilled in the art will recognize that the probe will contain asequence or “tail” that is not complementary to the single strandedamplicon members. In the case where the primer itself serves as thecapture label, at least a portion of the primer will be free tohybridize with a nucleic acid on a solid phase because the probe isselected such that it is not fully complementary to the primer sequence.

[0121] Generally, probe/single stranded amplicon member complexes can bedetected using techniques commonly employed to perform heterogeneousimmunoassays. Preferably, in this embodiment, detection is performedaccording to the protocols used by the commercially available AbbottLCx® instrumentation (Abbott Laboratories, Abbott Park, Ill.).

[0122] The primers and probes disclosed herein are useful in typical PCRassays, wherein the test sample is contacted with a pair of primers,amplification is performed, the hybridization probe is added, anddetection is performed.

[0123] Another method provided by the present invention comprisescontacting a test sample with a plurality of polynucleotides, wherein atleast one polynucleotide is a PS133 molecule as described herein,hybridizing the test sample with the plurality of polynucleotides anddetecting hybridization complexes. Hybridization complexes areidentified and quantitated to compile a profile which is indicative ofprostate tissue disease, such as prostate cancer. Expressed RNAsequences may further be detected by reverse transcription andamplification of the DNA product by procedures well-known in the art,including polymerase chain reaction (PCR).

[0124] Drug Screening and Gene Therapy.

[0125] The present invention also encompasses the use of gene therapymethods for the introduction of anti-sense PS133 derived molecules, suchas polynucleotides or oligonucleotides of the present invention, intopatients with conditions associated with abnormal expression ofpolynucleotides related to a prostate tissue disease or condition,especially prostate cancer. These molecules, including antisense RNA andDNA fragments and ribozymes, are designed to inhibit the translation ofPS133-mRNA, and may be used therapeutically in the treatment ofconditions associated with altered or abnormal expression of PS133polynucleotide.

[0126] Alternatively, the oligonucleotides described above can bedelivered to cells by procedures known in the art such that theanti-sense RNA or DNA may be expressed in vivo to inhibit production ofa PS133 polypeptide in the manner described above. Antisense constructsto a PS133 polynucleotide, therefore, reverse the action of PS133transcripts and may be used for treating prostate tissue diseaseconditions, such as prostate cancer. These antisense constructs may alsobe used to treat tumor metastases.

[0127] The present invention also provides a method of screening aplurality of compounds for specific binding to PS133 polypeptide(s), orany fragment thereof, to identify at least one compound whichspecifically binds the PS133 polypeptide. Such a method comprises thesteps of providing at least one compound; combining the PS133polypeptide with each compound under suitable conditions for a timesufficient to allow binding; and detecting the PS133 polypeptide bindingto each compound.

[0128] The polypeptide or peptide fragment employed in such a test mayeither be free in solution, affixed to a solid support, borne on a cellsurface or located intracellularly. One method of screening utilizeseukaryotic or prokaryotic host cells which are stably transfected withrecombinant nucleic acids which can express the polypeptide or peptidefragment. A drug, compound, or any other agent may be screened againstsuch transfected cells in competitive binding assays. For example, theformation of complexes between a polypeptide and the agent being testedcan be measured in either viable or fixed cells.

[0129] The present invention thus provides methods of screening fordrugs, compounds, or any other agent which can be used to treat diseasesassociated with PS133. These methods comprise contacting the agent witha polypeptide or fragment thereof and assaying for either the presenceof a complex between the agent and the polypeptide, or for the presenceof a complex between the polypeptide and the cell. In competitivebinding assays, the polypeptide typically is labeled. After suitableincubation, free (or uncomplexed) polypeptide or fragment thereof isseparated from that present in bound form, and the amount of free oruncomplexed label is used as a measure of the ability of the particularagent to bind to the polypeptide or to interfere with thepolypeptide/cell complex.

[0130] The present invention also encompasses the use of competitivescreening assays in which neutralizing antibodies capable of bindingpolypeptide specifically compete with a test agent for binding to thepolypeptide or fragment thereof. In this manner, the antibodies can beused to detect the presence of any polypeptide in the test sample whichshares one or more antigenic determinants with a PS133 polypeptide asprovided herein.

[0131] Another technique for screening provides high throughputscreening for compounds having suitable binding affinity to at least onepolypeptide of PS133 disclosed herein. Briefly, large numbers ofdifferent small peptide test compounds are synthesized on a solid phase,such as plastic pins or some other surface. The peptide test compoundsare reacted with polypeptide and washed. Polypeptide thus bound to thesolid phase is detected by methods well-known in the art. Purifiedpolypeptide can also be coated directly onto plates for use in thescreening techniques described herein. In addition, non-neutralizingantibodies can be used to capture the polypeptide and immobilize it onthe solid support. See, for example, EP 84/03564, published on Sep. 13,1984, which is incorporated herein by reference.

[0132] The goal of rational drug design is to produce structural analogsof biologically active polypeptides of interest or of the smallmolecules including agonists, antagonists, or inhibitors with which theyinteract. Such structural analogs can be used to design drugs which aremore active or stable forms of the polypeptide or which enhance orinterfere with the function of a polypeptide in vivo. J. Hodgson,Bio/Technology 9:19-21 (1991), incorporated herein by reference.

[0133] For example, in one approach, the three-dimensional structure ofa polypeptide, or of a polypeptide-inhibitor complex, is determined byx-ray crystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide may be gained by modeling basedon the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous polypeptide-likemolecules or to identify efficient inhibitors

[0134] Useful examples of rational drug design may include moleculeswhich have improved activity or stability as shown by S. Braxton et al.,Biochemistry 31:7796-7801 (1992), or which act as inhibitors, agonists,or antagonists of native peptides as shown by S. B. P. Athauda et al.,J. Biochem. (Tokyo) 113 (6):742-746 (1993), incorporated herein byreference.

[0135] The present invention is also directed to antagonists andinhibitors of the PS133 polypeptides of the present invention. Theantagonists and inhibitors are those which inhibit or eliminate thefunction of a PS133 polypeptide. Thus, for example, an antagonist maybind to the PS133 polypeptide of SEQUENCE ID NO 24, or fragment thereof,and inhibit or eliminate the function thereof. The antagonist, forexample, could be a peptide, antibody or oligonucleotide directedagainst the PS133 polypeptide of SEQUENCE ID NO 24, or fragment thereof.An example of an inhibitor is a small molecule inhibitor whichinactivates the PS133 polypeptide of SEQUENCE ID NO 24 by binding to andoccupying a catalytic site, thereby making the catalytic siteinaccessible to a substrate and preventing the biological activity ofthe PS133 polypeptide. Examples of small molecule inhibitors include,but are not limited to, small peptides or peptide-like molecules ornon-peptide protease inhibitors. These antagonists and inhibitors maythus be used to treat prostatic disease, such as benign prostatichypertrophy or prostatic cancer, by preventing a PS133 polypeptide fromfunctioning to catalyze the hydrolysis of its substrate.

[0136] It is also conceivable that a clinical condition exists whereinthe proteolytic activity of PS133 polypeptide is deficient, leading toan undesired medical situation. In this case, agonists of the PS133polypeptide are expected to be clinically useful. An agonist is acompound which might bind to an auxiliary site distal from the catalyticsite of the prostate protease and serve to activate the enzyme to ahigher state of function than in the situation without the agonistcompound. In this way, an agonist compound may be used to treat diseaseresulting from insufficient biological action of the PS133 polypeptide.

[0137] Compounds, agents or drugs which serve as antagonists, inhibitorsor agonists of PS133 polypeptides may be employed in a composition witha pharmaceutically acceptable carrier, including but not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol andcombinations thereof. Administration of protease inhibitors ispreferably by oral dose, but may also be intraperitoneal, subcutaneous,intravenous, intratracheal, sublingual, intranasal, rectal, transdermal.A direct application of the antagonists, agonists, or inhibitors, to thediseased prostate gland via injection into or near the prostate may alsobe employed.

[0138] A small molecule drug or compound may function by blocking orotherwise antagonizing the action of a PS133 polypeptide, or mayfunction as an agonist of an interaction between a PS133 polypeptide anda receptor. A therapeutically effective dose of such a drug or compoundcan be administered to a patient suffering from prostate disease, suchas prostate cancer, in the context of a therapeutic regimen that issufficient to cure, partially arrest, or detectably slow the progressionof the disease and its complications. The dosage of the drug or compoundmay be approximately 0.1-1000 mg per adult human, and more preferably,approximately 1-500 mg per adult human. The route of administration of asmall molecule drug or compound against a PS133 polypeptide may be anyroute which effectively transports the active compound to theappropriate or desired site of action, such as oral or parenteral, e.g.rectal, transdermal, depot, subcutaneous, intravenous, intramuscular orintranasal.

[0139] In order to be in a form that can be administered to a patient,one or more of the above-described drugs, agents or compounds (for usein the treatment of PS133-related disease) may be combined with one ormore non-toxic pharmaceutically acceptable carriers, diluents oradjuvants; other auxiliary agents if necessary; and, if desired, otheractive ingredients. Forms suitable for oral use include, but are notlimited to, tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsions, hard or soft capsules, orsyrups or elixirs, and may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions. Othercompositions for oral administration include spray compositions whichmay be prepared by known methods and which comprise one or more activecompounds. The spray compositions can also contain a suitablepropellant.

[0140] Pharmaceutical compositions for parenteral administration byinjection of a PS133-related drug, agent, or compound, include sterileaqueous or non-aqueous solutions and suspensions or emulsions, in anontoxic parenterally-acceptable diluent or solvent. These compositionsmay also include adjuvants such as wetting, preserving, emulsifying anddispersing agents. They may be sterilized by one of several knownmethods or manufactured in the form of sterile solid compositions whichcan be dissolved in sterile water or some other sterile injectablemedium immediately before use.

[0141] Pharmaceutical compositions may also be administered in the formof suppositories for rectal administration. These compositions can beprepared by mixing an appropriate drug, agent or compound with asuitable non-irritating excipient which is solid at ordinarytemperatures but liquid at rectal temperatures, and will therefore meltin the rectum to release the therapeutically active ingredient.

[0142] Formulations containing PS133 drugs, agents or compounds may beformulated so as to provide quick, sustained, or delayed release of theactive ingredient after administration to the patient by employingprocedures well known in the art.

[0143] Active compounds to PS133 may be suitable for administration toan animal. Such animals include domestic animals, for example livestock,laboratory animals, and household pets, and non-domestic animals such aswildlife. More preferably, the animal is a vertebrate. Most preferably,a drug to PS133 is administered to a mammal. It is especially preferredthat the animal is a domestic mammal or a human. The most preferredmammal is a human. For such purposes, a PS133 drug, agent or compoundmay be administered as a food or feed additive.

[0144] The present invention also relates to an assay for identifyingthe above-mentioned small molecule inhibitors which are specific to aPS133 protease and prevent it from functioning. Either natural proteinsubstrates or synthetic peptides can be used to assess proteolyticactivity of the protease molecule, and the ability of inhibitors toprevent this activity provides the basis for a screen to identify acompound that has therapeutic activity in prostatic disorders.

[0145] It also is possible to isolate a target-specific antibodyselected by an assay as described hereinabove, and then to determine itscrystal structure. In principle this approach yields a pharmacophoreupon which subsequent drug design can be based. It further is possibleto bypass protein crystallography altogether by generatinganti-idiotypic antibodies (“anti-ids”) to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of the anti-id is an analog of the original receptor.The anti-id then can be used to identify and isolate peptides from banksof chemically or biologically produced peptides. The isolated peptidesthen can act as the pharmacophore (that is, a prototype pharmaceuticaldrug).

[0146] A sufficient amount of a recombinant polypeptide of the presentinvention may be made available to perform analytical studies such asX-ray crystallography. In addition, knowledge of the polypeptide aminoacid sequence which is derivable from the nucleic acid sequence providedherein will provide guidance to those employing computer modelingtechniques in place of, or in addition to, x-ray crystallography.

[0147] Antibodies specific to a PS133 polypeptide (e.g., anti-PS133antibodies) further may be used to inhibit the biological action of thepolypeptide by binding to the polypeptide. In this manner, theantibodies may be used in therapy, for example, to treat prostate tissuediseases including prostate cancer and its metastases.

[0148] Further, such antibodies can detect the presence or absence of aPS133 polypeptide in a test sample and, therefore, are useful asdiagnostic markers for the diagnosis of a prostate tissue disease orcondition, especially prostate cancer. Such antibodies may also functionas a diagnostic marker for prostate tissue disease conditions, such asprostate cancer. The present invention also is directed to antagonistsand inhibitors of the polypeptides of the present invention. Theantagonists and inhibitors are those which inhibit or eliminate thefunction of the polypeptide. Thus, for example, an antagonist may bindto a polypeptide of the present invention and inhibit or eliminate itsfunction. The antagonist, for example, could be an antibody against thepolypeptide which eliminates the activity of a PS133 polypeptide bybinding a PS133 polypeptide, or in some cases the antagonist may be anoligonucleotide. Examples of small molecule inhibitors include, but arenot limited to, small peptides or peptide-like molecules.

[0149] The antagonists and inhibitors may be employed as a compositionwith a pharmaceutically acceptable carrier including, but not limitedto, saline, buffered saline, dextrose, water, glycerol, ethanol andcombinations thereof. Administration of PS133 polypeptide inhibitors ispreferably systemic. The present invention also provides an antibodywhich inhibits the action of such a polypeptide.

[0150] The antibody may be a monoclonal antibody that demonstratesbinding specificity for PS133, and functions as a therapeutic agent byblocking or otherwise antagonizing the action of PS133 in diseasedprostate cells, such as prostate tumor cells, or may function as anagonist of an interaction between PS133 and a receptor of prostate tumorcells. Such an antibody may be administered in vivo to a patient alreadysuffering from prostate disease, such as prostate cancer, in atherapeutically effective or efficacious dose to cure, partially arrest,or detectably slow the progression of the disease and its complications.The amounts effective for this use will depend upon the severity of theprostate disease, the general state of the patient, and the route ofadministration and combination with other active agents to prostatedisease, such as prostate cancer.

[0151] Pharmaceutical compositions containing the monoclonal antibodyalso can be administered either systemically, by intravenous infusion,or locally, by injection. The composition may include pharmaceuticalcarriers or diluents. The diluent is selected so as not to affect thebiological activity of the composition. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0152] For the destruction of disease cells, such as prostate tumorcells, it can be advantageous to conjugate the PS133-specific monoclonalantibody to another molecule prior to in vivo administration. Forexample, the monoclonal antibody can be joined to a cytotoxin, achemotherapeutic agent, a radioisotope, or other modulator of cellularactivity, whereby binding of the monoclonal antibody conjugate toprostate tumor cells will result in tumor cell death. For example, anumber of protein toxins are well known in the art including ricin,diphtheria, gelonin, Pseudomonas toxin, and arbrin. Chemotherapeuticagents include, but are not limited to, for example, methotrexate,daunorubicin, and doxorubicin. Radioisotopes include, but are notlimited to, yttrium-90, phosphorus-32, lead-212, iodine-131, andpalladium-109.

[0153] Antisense technology can be used to reduce gene expressionthrough triple-helix formation or antisense DNA or RNA, both of whichmethods are based on binding of a polynucleotide to DNA or RNA. Forexample, the 5′ coding portion of the polynucleotide sequence, whichencodes for the polypeptide of the present invention, is used to designan antisense RNA oligonucleotide of from 10 to 40 base pairs in length.A DNA oligonucleotide is designed to be complementary to a region of thegene involved in transcription, thereby preventing transcription and theproduction of the PS133 polypeptide. For triple helix, see, for example,Lee et al., Nuc. Acids Res. 6:3073 (1979); Cooney et al., Science241:456 (1988); and Dervan et al., Science 251:1360 (1991) The antisenseoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofa mRNA molecule into the PS133 polypeptide. For antisense, see, forexample, Okano, J. Neurochem. 56:560 (1991); and “Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression,” CRC Press, Boca Raton, Fla.(1988). Antisense oligonucleotides act with greater efficacy whenmodified to contain artificial intemucleotide linkages which render themolecule resistant to nucleolytic cleavage. Such artificialinternucleotide linkages include, but are not limited to,methylphosphonate, phosphorothiolate and phosphoroamydate intemucleotidelinkages.

[0154] Administration of an antisense PS133 oligonucleotide to anindividual may be orally or by subcutaneous, intramuscular,intraperitoneal, or intravenous injection. A typical injectablecomposition comprises a pharmaceutically acceptable solvent or diluent,and other suitable, physiologic compounds. For example, the compositionmay contain an antisense PS133 oligonucleotide and about 10 mg/ml ofhuman serum albumin in a phosphate buffer containing NaCl. Otherpharmaceutically acceptable excipients include, non-aqueous or aqueoussolutions and non-toxic compositions including salts, preservatives,buffers and the like. The pH and exact concentration of the variouscomponents are adjusted according to routine skills in the art.

[0155] An antisense PS133 oligonucleotide may also be administered byinjection as an oily suspension. Suitable lipophilic solvents orvehicles include fatty oils, synthetic fatty acid esters, or any one ofa number of sterols. A preferred sterol is cholesterol. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension and also stabilizers.

[0156] An alternative formulation for antisense oligonucleotideadministration involves liposomes. Antisense oligonucleotides can beencapsulated within liposomes using standard techniques. A variety ofdifferent liposome compositions and methods for synthesis are known tothose of skill in the art. In general, the dosage of administeredliposome-encapsulated antisense PS133 oligonucleotide will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous history. Dose ranges forparticular formulations can be determined by using a suitable animalmodel.

[0157] Recombinant Technology.

[0158] The present invention provides host cells and expression vectorscomprising PS133 polynucleotides of the present invention and methodsfor the production of the polypeptide(s) they encode. Such methodscomprise culturing the host cells under conditions suitable for theexpression of the PS133 polynucleotide and recovering the PS133polypeptide from the cell culture.

[0159] The present invention also provides vectors which include PS133polynucleotides of the present invention, host cells which aregenetically engineered with vectors of the present invention and theproduction of polypeptides of the present invention by recombinanttechniques.

[0160] Host cells are genetically engineered (transfected, transduced ortransformed) with the vectors of this invention which may be cloningvectors or expression vectors. The vector may be in the form of aplasmid, a viral particle, a phage, etc. The engineered host cells canbe cultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transfected cells, or amplifying PS133gene(s). The culture conditions, such as temperature, pH and the like,are those previously used with the host cell selected for expression,and will be apparent to the ordinarily skilled artisan.

[0161] The polynucleotides of the present invention may be employed forproducing a polypeptide by recombinant techniques. Thus, thepolynucleotide sequence may be included in any one of a variety ofexpression vehicles, in particular vectors or plasmids for expressing apolypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; yeast plasmids; vectors derived from combinations of plasmidsand phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virusand pseudorabies. However, any other plasmid or vector may be used solong as it is replicable and viable in the host.

[0162] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted intoappropriate restriction endonuclease sites by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art. The DNA sequence in the expression vector isoperatively linked to an appropriate expression control sequencers)(promoter) to direct mRNA synthesis. Representative examples of suchpromoters include, but are not limited to, the LTR or the SV40 promoter,the E. coli lac or trp, the phage lambda P sub L promoter and otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. The expression vector also contains aribosome binding site for translation initiation and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression. In addition, the expression vectors preferablycontain a gene to provide a phenotypic trait for selection oftransfected host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0163] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transfect an appropriate host to permit the host toexpress the protein. As representative examples of appropriate hosts,there may be mentioned: bacterial cells, such as E. coli, Salmonellatyphimurium; Streptomyces sp; fungal cells, such as yeast; insect cellssuch as Drosophila and Sf9; animal cells such as CHO, COS or Bowesmelanoma; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings provided herein.

[0164] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises-regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art and are commercially available. The following vectorsare provided by way of example. Bacterial: pINCY (Incyte PharmaceuticalsInc., Palo Alto, Calif.), pSPORT1 (Life Technologies, Gaithersburg,Md.), pQE70, pQE60, pQE-9 (Qiagen) pBs, phagescript, psiX174,pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene);pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic:pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL(Pharmacia). However, any other plasmid or vector may be used as long asit is replicable and viable in the host.

[0165] Plasmid pINCY is generally identical to the plasmid pSPORT1(available from Life Technologies, Gaithersburg, Md.) with the exceptionthat it has two modifications in the polylinker (multiple cloning site).These modifications are (1) it lacks a HindIII restriction site and (2)its EcoRI restriction site lies at a different location. pINCY iscreated from pSPORT1 by cleaving pSPORT1 with both HindIII and EcoRI andreplacing the excised fragment of the polylinker with synthetic DNAfragments (SEQUENCE ID NO 9 and SEQUENCE ID NO 10). This replacement maybe made in any manner known to those of ordinary skill in the art. Forexample, the two nucleotide sequences, SEQUENCE ID NO 9 and SEQUENCE IDNO 10, may be generated synthetically with 5′ terminal phosphates, mixedtogether, and then ligated under standard conditions for performingstaggered end ligations into the pSPORT1 plasmid cut with HindIII andEcoRI. Suitable host cells (such as E. coli DH5∝ cells) then aretransfected with the ligated DNA and recombinant clones are selected forampicillin resistance. Plasmid DNA then is prepared from individualclones and subjected to restriction enzyme analysis or DNA sequencing inorder to confirm the presence of insert sequences in the properorientation. Other cloning strategies known to the ordinary artisan alsomay be employed.

[0166] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lac, lacZ, T3, SP6, T7, gpt, lambda P sub R,P sub L and trp. Eukaryotic promoters include cytomegalovirus (CMV)immediate early, herpes simplex virus (HSV) thymidine kinase, early andlate SV40, LTRs from retroviruses and mouse metallothionein-I. Selectionof the appropriate vector and promoter is well within the level ofordinary skill in the art.

[0167] In a further embodiment, the present invention provides hostcells containing the above-described construct. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (L. Davis et al., “BasicMethods in Molecular Biology,” 2nd edition, Appleton and Lang, ParamountPublishing, East Norwalk, Conn. (1994)).

[0168] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0169] Recombinant proteins can be expressed in mammalian cells, yeast,bacteria, or other cells, under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (Cold SpringHarbor, N.Y., 1989), which is hereby incorporated by reference.

[0170] Transcription of a DNA encoding the polypeptide(s) of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin (bp 100 to 270), a cytomegalovirus early promoterenhancer, a polyoma enhancer on the late side of the replication originand adenovirus enhancers.

[0171] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transfection of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0172] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransfection include E. coli, Bacillus subtilis, Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces andStaphylococcus, although, others may also be employed as a routinematter of choice.

[0173] Useful expression vectors for bacterial use comprise a selectablemarker and bacterial origin of replication derived from plasmidscomprising genetic elements of the well-known cloning vector pBR322(ATCC 37017). Other vectors include but are not limited to PKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis.). These pBR322 “backbone” sections are combined with anappropriate promoter and the structural sequence to be expressed.

[0174] Following transfection of a suitable host and growth of the hostto an appropriate cell density, the selected promoter is derepressed byappropriate means (e.g., temperature shift or chemical induction), andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell-known to one of ordinary skill in the art.

[0175] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts described byGluzman, Cell 23:175 (1981), and other cell lines capable of expressinga compatible vector, such as the C127, HEK-293, 3T3, CHO, HeLa and BHKcell lines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer and also any necessaryribosome binding sites, polyadenylation sites, splice donor and acceptorsites, transcriptional termination sequences and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 viralgenome, for example, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements. Representative, useful vectors include pRc/CMV andpcDNA3 (available from Invitrogen, San Diego, Calif.).

[0176] PS133 polypeptides are recovered and purified from recombinantcell cultures by known methods including affinity chromatography,ammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, hydroxyapatite chromatography orlectin chromatography. It is preferred to have low concentrations(approximately 0.1-5 mM) of calcium ion present during purification(Price et al., J. Biol. Chem. 244:917 (1969)). Protein refolding stepscan be used, as necessary, in completing configuration of thepolypeptide. Finally, high performance liquid chromatography (HPLC) canbe employed for final purification steps.

[0177] Thus, polypeptides of the present invention may be naturallypurified products expressed from a high expressing cell line, or aproduct of chemical synthetic procedures, or produced by recombinanttechniques from a prokaryotic or eukaryotic host (for example, bybacterial, yeast, higher plant, insect and mammalian cells in culture).Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated withmammalian or other eukaryotic carbohydrates or may be non-glycosylated.The polypeptides of the invention may also include an initial methionineamino acid residue.

[0178] The starting plasmids can be constructed from available plasmidsin accord with published, known procedures. In addition, equivalentplasmids to those described are known in the art and will be apparent toone of ordinary skill in the art.

[0179] The following is the general procedure for the isolation andanalysis of cDNA clones. In a particular embodiment disclosed herein,mRNA was isolated from prostate tissue and used to generate the cDNAlibrary. Prostate tissue was obtained from patients by surgicalresection and was classified as tumor or non-tumor tissue by apathologist.

[0180] The cDNA inserts from random isolates of the prostate tissuelibraries were sequenced in part, analyzed in detail as set forth in theExamples and are disclosed in the Sequence Listing as SEQUENCE ID NOS1-6. The consensus sequence of these inserts is presented as SEQUENCE IDNO 7. These polynucleotides may contain an entire open reading framewith or without associated regulatory sequences for a particular gene,or they may encode only a portion of the gene of interest. This isattributed to the fact that many genes are several hundred and sometimesseveral thousand, bases in length and, with current technology, cannotbe cloned in their entirety because of vector limitations, incompletereverse transcription of the first strand, or incomplete replication ofthe second strand. Contiguous, secondary clones containing additionalnucleotide sequences may be obtained using a variety of methods known tothose of skill in the art.

[0181] Methods for DNA sequencing are well known in the art.Conventional enzymatic methods employ DNA polymerase, Klenow fragment,Sequenase (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest. Methods have been developed for the use of bothsingle-stranded and double-stranded templates. The chain terminationreaction products may be electrophoresed on urea/polyacrylamide gels anddetected either by autoradiography (for radionucleotide labeledprecursors) or by fluorescence (for fluorescent-labeled precursors).Recent improvements in mechanized reaction preparation, sequencing andanalysis using the fluorescent detection method have permitted expansionin the number of sequences that can be determined per day using machinessuch as the Applied Biosystems 377 DNA Sequencers (Applied Biosystems,Foster City, Calif.).

[0182] The reading frame of the nucleotide sequence can be ascertainedby several types of analyses. First, reading frames contained within thecoding sequence can be analyzed for the presence of start codon ATG andstop codons TGA, TAA or TAG. Typically, one reading frame will continuethroughout the major portion of a cDNA sequence while other readingframes tend to contain numerous stop codons. In such cases, readingframe determination is straightforward. In other more difficult cases,further analysis is required.

[0183] Algorithms have been created to analyze the occurrence ofindividual nucleotide bases at each putative codon triplet. See, forexample J. W. Fickett, Nuc Acids Res 10:5303 (1982). Coding DNA forparticular organisms (bacteria, plants and animals) tends to containcertain nucleotides within certain triplet periodicities, such as asignificant preference for pyrimidines in the third codon position.These preferences have been incorporated into widely available softwarewhich can be used to determine coding potential (and frame) of a givenstretch of DNA. The algorithm-derived information combined withstart/stop codon information can be used to determine proper frame witha high degree of certainty. This, in turn, readily permits cloning ofthe sequence in the correct reading frame into appropriate expressionvectors.

[0184] The nucleic acid sequences disclosed herein may be joined to avariety of other polynucleotide sequences and vectors of interest bymeans of well-established recombinant DNA techniques. See J. Sambrook etal., supra. Vectors of interest include cloning vectors, such asplasmids, cosmids, phage derivatives, phagemids, as well as sequencing,replication and expression vectors, and the like. In general, suchvectors contain an origin of replication functional in at least oneorganism, convenient restriction endonuclease digestion sites andselectable markers appropriate for particular host cells. The vectorscan be transferred by a variety of means known to those of skill in theart into suitable host cells which then produce the desired DNA, RNA orpolypeptides.

[0185] Occasionally, sequencing or random reverse transcription errorswill mask the presence of the appropriate open reading frame orregulatory element. In such cases, it is possible to determine thecorrect reading frame by attempting to express the polypeptide anddetermining the amino acid sequence by standard peptide mapping andsequencing techniques. See, F. M. Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y. (1989).Additionally, the actual reading frame of a given nucleotide sequencemay be determined by transfection of host cells with vectors containingall three potential reading frames. Only those cells with the nucleotidesequence in the correct reading frame will produce a peptide of thepredicted length.

[0186] The nucleotide sequences provided herein have been prepared bycurrent, state-of-the-art, automated methods and, as such, may containunidentified nucleotides. These will not present a problem to thoseskilled in the art who wish to practice the invention. Several methodsemploying standard recombinant techniques, described in J. Sambrook(supra) or periodic updates thereof, may be used to complete the missingsequence information. The same techniques used for obtaining a fulllength sequence, as described herein, may be used to obtain nucleotidesequences.

[0187] Expression of a particular cDNA may be accomplished by subcloningthe cDNA into an appropriate expression vector and transfecting thisvector into an appropriate expression host. The cloning vector used forthe generation of the prostate tissue cDNA library can be used fortranscribing mRNA of a particular cDNA and contains a promoter forbeta-galactosidase, an amino-terminal met and the subsequent seven aminoacid residues of beta-galactosidase. Immediately following these eightresidues is an engineered bacteriophage promoter useful for artificialpriming and transcription, as well as a number of unique restrictionsites, including EcoRI, for cloning. The vector can be transfected intoan appropriate host strain of E. coli.

[0188] Induction of the isolated bacterial strain withisopropylthiogalactoside (IPTG) using standard methods will produce afusion protein which contains the first seven residues ofbeta-galactosidase, about 15 residues of linker and the peptide encodedwithin the cDNA. Since cDNA clone inserts are generated by anessentially random process, there is one chance in three that theincluded cDNA will lie in the correct frame for proper translation. Ifthe cDNA is not in the proper reading frame, the correct frame can beobtained by deletion or insertion of an appropriate number of bases bywell known methods including in vitro mutagenesis, digestion withexonuclease III or mung bean nuclease, or oligonucleotide linkerinclusion.

[0189] The cDNA can be shuttled into other vectors known to be usefulfor expression of protein in specific hosts. Oligonucleotide primerscontaining cloning sites and segments of DNA sufficient to hybridize tostretches at both ends of the target cDNA, can be synthesized chemicallyby standard methods. These primers can then be used to amplify thedesired gene segments by PCR. The resulting new gene segments can bedigested with appropriate restriction enzymes under standard conditionsand isolated by gel electrophoresis. Alternately, similar gene segmentscan be produced by digestion of the cDNA with appropriate restrictionenzymes and filling in the missing gene segments with chemicallysynthesized oligonucleotides. Segments of the coding sequence from morethan one gene can be ligated together and cloned in appropriate vectorsto optimize expression of recombinant sequence.

[0190] Suitable expression hosts for such chimeric molecules include,but are not limited to, mammalian cells such as Chinese Hamster Ovary(CHO) and human embryonic kidney (HEK) 293 cells, insect cells such asSf9 cells, yeast cells such as Saccharomyces cerevisiae and bacteriasuch as E. coli. For each of these cell systems, a useful expressionvector may also include an origin of replication to allow propagation inbacteria and a selectable marker such as the beta-lactamase antibioticresistance gene to allow selection in bacteria. In addition, the vectorsmay include a second selectable marker, such as the neomycinphosphotransferase gene, to allow selection in transfected eukaryotichost cells. Vectors for use in eukaryotic expression hosts may requirethe addition of 3′ poly A tail if the sequence of interest lacks poly A.

[0191] Additionally, the vector may contain promoters or enhancers whichincrease gene expression. Such promoters are host specific and include,but are not limited to, MMTV, SV40, or metallothionine promoters for CHOcells; trp, lac, tac or T7 promoters for bacterial hosts; or alphafactor, alcohol oxidase or PGH promoters for yeast. Adenoviral vectorswith or without transcription enhancers, such as the rous sarcoma virus(RSV) enhancer, may be used to drive protein expression in mammaliancell lines. Once homogeneous cultures of recombinant cells are obtained,large quantities of recombinantly produced protein can be recovered fromthe conditioned medium and analyzed using chromatographic methods wellknown in the art. An alternative method for the production of largeamounts of secreted protein involves the transfection of mammalianembryos and the recovery of the recombinant protein from milk producedby transgenic cows, goats, sheep, etc. Polypeptides and closely relatedmolecules may be expressed recombinantly in such a way as to facilitateprotein purification. One approach involves expression of a chimericprotein which includes one or more additional polypeptide domains notnaturally present on human polypeptides. Such purification-facilitatingdomains include, but are not limited to, metal-chelating peptides suchas histidine-tryptophan domains that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle, Wash.). The inclusion of acleavable linker sequence such as Factor XA or enterokinase fromInvitrogen (San Diego, Calif.) between the polypeptide sequence and thepurification domain may be useful for recovering the polypeptide.

[0192] Immunoassays.

[0193] PS133 polypeptides, including fragments, derivatives, and analogsthereof, or cells expressing such polypeptides, can be utilized in avariety of assays, many of which are described herein, for the detectionof antibodies to prostate tissue. They also can be used as immunogens toproduce antibodies. These antibodies can be, for example, polyclonal ormonoclonal antibodies, chimeric, single chain and humanized antibodies,as well as Fab fragments, or the product of an Fab expression library.Various procedures known in the art may be used for the production ofsuch antibodies and fragments.

[0194] For example, antibodies generated against a polypeptidecomprising a sequence of the present invention can be obtained by directinjection of the polypeptide into an animal or by administering thepolypeptide to an animal such as a mouse, rabbit, goat or human. Amouse, rabbit or goat is preferred. The polypeptide is selected from thegroup consisting of SEQUENCE ID NO 24, SEQUENCE ID NO 25, SEQUENCE ID NO26, SEQUENCE ID NO 27, and fragments thereof. The antibody so obtainedthen will bind the polypeptide itself. In this manner, even a sequenceencoding only a fragment of the polypeptide can be used to generateantibodies that bind the native polypeptide. Such antibodies then can beused to isolate the polypeptide from test samples such as tissuesuspected of containing that polypeptide. For preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples include thehybridoma technique as described by Kohler and Milstein, Nature256:495-497 (1975), the trioma technique, the human B-cell hybridomatechnique as described by Kozbor et al., Immun. Today 4:72 (1983) andthe EBV-hybridoma technique to produce human monoclonal antibodies asdescribed by Cole, et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc, New York, N.Y., pp. 77-96 (1985). Techniquesdescribed for the production of single chain antibodies can be adaptedto produce single chain antibodies to immunogenic polypeptide productsof this invention. See, for example, U.S. Pat. No. 4,946,778, which isincorporated herein by reference.

[0195] Various assay formats may utilize the antibodies of the presentinvention, including “sandwich” immunoassays and probe assays. Forexample, the antibodies of the present invention, or fragments thereof,can be employed in various assay systems to determine the presence, ifany, of PS133 antigen in a test sample. For example, in a first assayformat, a polyclonal or monoclonal antibody or fragment thereof, or acombination of these antibodies, which has been coated on a solid phase,is contacted with a test sample, to form a first mixture. This firstmixture is incubated for a time and under conditions sufficient to formantigen/antibody complexes. Then, an indicator reagent comprising amonoclonal or a polyclonal antibody or a fragment thereof, or acombination of these antibodies,to which a signal generating compoundhas been attached, is contacted with the antigen/antibody complexes toform a second mixture. This second mixture then is incubated for a timeand under conditions sufficient to form antibody/antigen/antibodycomplexes. The presence of PS133 antigen in the test sample and capturedon the solid phase, if any, is determined by detecting the measurablesignal generated by the signal generating compound. The amount of PS133antigen present in the test sample is proportional to the signalgenerated.

[0196] In an alternative assay format, a mixture is formed bycontacting: (1) a polyclonal antibody, monoclonal antibody, or fragmentthereof, which specifically binds to PS133 antigen, or a combination ofsuch antibodies bound to a solid support; (2) the test sample; and (3)an indicator reagent comprising a monoclonal antibody, polyclonalantibody, or fragment thereof, which specifically binds to a differentPS133 antigen (or a combination of these antibodies) to which a signalgenerating compound is attached. This mixture is incubated for a timeand under conditions sufficient to form antibody/antigen/antibodycomplexes. The presence, if any, of PS133 antigen present in the testsample and captured on the solid phase is determined by detecting themeasurable signal generated by the signal generating compound. Theamount of PS133 antigen present in the test sample is proportional tothe signal generated.

[0197] In another assay format, one or a combination of at least twomonoclonal antibodies of the invention can be employed as a competitiveprobe for the detection of antibodies to PS133 antigen. For example,PS133 polypeptides such as the recombinant antigens disclosed herein,either alone or in combination, are coated on a solid phase. A testsample suspected of containing antibody to PS133 antigen then isincubated with an indicator reagent comprising a signal generatingcompound and at least one monoclonal antibody of the invention for atime and under conditions sufficient to form antigen/antibody complexesof either the test sample and indicator reagent bound to the solid phaseor the indicator reagent bound to the solid phase. The reduction inbinding of the monoclonal antibody to the solid phase can bequantitatively measured.

[0198] In yet another detection method, each of the monoclonal orpolyclonal antibodies of the present invention can be employed in thedetection of PS133 antigens in tissue sections, as well as in cells, byimmunohistochemical analysis. Cytochemical analysis wherein theseantibodies are labeled directly (with, for example, fluorescein,colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) orare labeled by using secondary labeled anti-species antibodies (withvarious labels as exemplified herein) to track the histopathology ofdisease also are within the scope of the present invention.

[0199] In addition, these monoclonal antibodies can be bound to matricessimilar to CNBr-activated Sepharose and used for the affinitypurification of specific PS133 polypeptides from cell cultures orbiological tissues such as to purify recombinant and native PS133proteins.

[0200] The monoclonal antibodies of the invention also can be used forthe generation of chimeric antibodies for therapeutic use, or othersimilar applications.

[0201] The monoclonal antibodies or fragments thereof can be providedindividually to detect PS133 antigens. Combinations of the monoclonalantibodies (and fragments thereof) provided herein also may be usedtogether as components in a mixture or “cocktail” of at least one PS133antibody of the invention, along with antibodies which specifically bindto other PS133 regions, each antibody having different bindingspecificities. Thus, this cocktail can include the monoclonal antibodiesof the invention which are directed to PS133 polypeptides disclosedherein and other monoclonal antibodies specific to other antigenicdeterminants of PS133 antigens or other related proteins.

[0202] The polyclonal antibody or fragment thereof which can be used inthe assay formats should specifically bind to a PS133 polypeptide orother PS133 polypeptides additionally used in the assay. The polyclonalantibody used preferably is of mammalian origin such as, human, goat,rabbit or sheep polyclonal antibody which binds PS133 polypeptide. Mostpreferably, the polyclonal antibody is of rabbit origin. The polyclonalantibodies used in the assays can be used either alone or as a cocktailof polyclonal antibodies. Since the cocktails used in the assay formatsare comprised of either monoclonal antibodies or polyclonal antibodieshaving different binding specificity to PS133 polypeptides, they areuseful for the detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, or determining thepredisposition to, diseases and conditions of the prostate, such asprostate cancer.

[0203] It is contemplated and within the scope of the present inventionthat PS133 antigen may be detectable in assays by use of a recombinantantigen as well as by use of a synthetic peptide or purified peptide,which peptide comprises an amino acid sequence of PS133. The amino acidsequence of such a polypeptide is selected from the group consisting ofSEQUENCE ID NO 24, SEQUENCE ID NO 25, SEQUENCE ID NO 26, SEQUENCE ID NO27, and fragments thereof. It also is within the scope of the presentinvention that different synthetic, recombinant or purified peptides,identifying different epitopes of PS133, can be used in combination inan assay for the detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, or determining thepredisposition to diseases and conditions of the prostate, such asprostate cancer. In this case, all of these peptides can be coated ontoone solid phase; or each separate peptide may be coated onto separatesolid phases, such as microparticles, and then combined to form amixture of peptides which can be later used in assays. Furthermore, itis contemplated that multiple peptides which define epitopes fromdifferent antigens may be used for the detection, diagnosis, staging,monitoring, prognosis, prevention or treatment of, or determining thepredisposition to, diseases and conditions of the prostate, such asprostate cancer. Peptides coated on solid phases or labeled withdetectable labels are then allowed to compete with those present in apatient sample (if any) for a limited amount of antibody. A reduction inbinding of the synthetic, recombinant, or purified peptides to theantibody (or antibodies) is an indication of the presence of PS133antigen in the patient sample. The presence of PS133 antigen indicatesthe presence of prostate tissue disease, especially prostate cancer, inthe patient. Variations of assay formats are known to those of ordinaryskill in the art and many are discussed herein below.

[0204] In another assay format, the presence of anti-PS133 antibodyand/or PS133 antigen can be detected in a simultaneous assay, asfollows. A test sample is simultaneously contacted with a capturereagent of a first analyte, wherein said capture reagent comprises afirst binding member specific for a first analyte attached to a solidphase and a capture reagent for a second analyte, wherein said capturereagent comprises a first binding member for a second analyte attachedto a second solid phase, to thereby form a mixture. This mixture isincubated for a time and under conditions sufficient to form capturereagent/first analyte and capture reagent/second analyte complexes.These so-formed complexes then are contacted with an indicator reagentcomprising a member of a binding pair specific for the first analytelabeled with a signal generating compound and an indicator reagentcomprising a member of a binding pair specific for the second analytelabeled with a signal generating compound to form a second mixture. Thissecond mixture is incubated for a time and under conditions sufficientto form capture reagent/first analyte/indicator reagent complexes andcapture reagent/second analyte/indicator reagent complexes. The presenceof one or more analytes is determined by detecting a signal generated inconnection with the complexes formed on either or both solid phases asan indication of the presence of one or more analytes in the testsample. In this assay format, recombinant antigens derived from theexpression systems disclosed herein may be utilized, as well asmonoclonal antibodies produced from the proteins derived from theexpression systems as disclosed herein. For example, in this assaysystem, PS133 antigen can be the first analyte. Such assay systems aredescribed in greater detail in EP Publication No. 0473065.

[0205] In yet other assay formats, the polypeptides disclosed herein maybe utilized to detect the presence of antibody against PS133 antigen intest samples. For example, a test sample is incubated with a solid phaseto which at least one polypeptide such as a recombinant protein orsynthetic peptide has been attached. The polypeptide is selected fromthe group consisting of SEQUENCE ID NO 24, SEQUENCE ID NO 25, SEQUENCEID NO 26, SEQUENCE ID NO 27, and fragments thereof. These are reactedfor a time and under conditions sufficient to form antigen/antibodycomplexes. Following incubation, the antigen/antibody complex isdetected. Indicator reagents may be used to facilitate detection,depending upon the assay system chosen. In another assay format, a testsample is contacted with a solid phase to which a recombinant proteinproduced as described herein is attached, and also is contacted with amonoclonal or polyclonal antibody specific for the protein, whichpreferably has been labeled with an indicator reagent. After incubationfor a time and under conditions sufficient for antibody/antigencomplexes to form, the solid phase is separated from the free phase, andthe label is detected in either the solid or free phase as an indicationof the presence of antibody against PS133 antigen. Other assay formatsutilizing the recombinant antigens disclosed herein are contemplated.These include contacting a test sample with a solid phase to which atleast one antigen from a first source has been attached, incubating thesolid phase and test sample for a time and under conditions sufficientto form antigen/antibody complexes, and then contacting the solid phasewith a labeled antigen, which antigen is derived from a second sourcedifferent from the first source. For example, a recombinant proteinderived from a first source such as E. coli is used as a capture antigenon a solid phase, a test sample is added to the so-prepared solid phase,and following standard incubation and washing steps as deemed orrequired, a recombinant protein derived from a different source (i.e.,non-E. coli) is utilized as a part of an indicator reagent whichsubsequently is detected. Likewise, combinations of a recombinantantigen on a solid phase and synthetic peptide in the indicator phasealso are possible. Any assay format which utilizes an antigen specificfor PS133 produced or derived from a first source as the capture antigenand an antigen specific for PS133 from a different second source iscontemplated. Thus, various combinations of recombinant antigens, aswell as the use of synthetic peptides, purified proteins and the like,are within the scope of this invention. Assays such as this and othersare described in U.S. Pat. No. 5,254,458, which enjoys common ownershipand is incorporated herein by reference.

[0206] Other embodiments which utilize various other solid phases alsoare contemplated and are within the scope of this invention. Forexample, ion capture procedures for immobilizing an immobilizablereaction complex with a negatively charged polymer (described in EPpublication 0326100 and EP publication No. 0406473), can be employedaccording to the present invention to effect a fast solution-phaseimmunochemical reaction. An immobilizable immune complex is separatedfrom the rest of the reaction mixture by ionic interactions between thenegatively charged poly-anion/immune complex and the previously treated,positively charged porous matrix and detected by using various signalgenerating systems previously described, including those described inchemiluminescent signal measurements as described in EPO Publication No.0 273,115.

[0207] Also, the methods of the present invention can be adapted for usein systems which utilize microparticle technology including automatedand semi-automated systems wherein the solid phase comprises amicroparticle (magnetic or non-magnetic). Such systems include thosedescribed in, for example, published EPO applications Nos. EP 0 425 633and EP 0 424 634, respectively.

[0208] The use of scanning probe microscopy (SPM) for immunoassays alsois a technology to which the monoclonal antibodies of the presentinvention are easily adaptable. In scanning probe microscopy,particularly in atomic force microscopy, the capture phase, for example,at least one of the monoclonal antibodies of the invention, is adheredto a solid phase and a scanning probe microscope is utilized to detectantigen/antibody complexes which may be present on the surface of thesolid phase. The use of scanning tunneling microscopy eliminates theneed for labels which normally must be utilized in many immunoassaysystems to detect antigen/antibody complexes. The use of SPM to monitorspecific binding reactions can occur in many ways. In one embodiment,one member of a specific binding partner (analyte specific substancewhich is the monoclonal antibody of the invention) is attached to asurface suitable for scanning. The attachment of the analyte specificsubstance may be by adsorption to a test piece which comprises a solidphase of a plastic or metal surface, following methods known to those ofordinary skill in the art. Or, covalent attachment of a specific bindingpartner (analyte specific substance) to a test piece which test piececomprises a solid phase of derivatized plastic, metal, silicon, or glassmay be utilized. Covalent attachment methods are known to those skilledin the art and include a variety of means to irreversibly link specificbinding partners to the test piece. If the test piece is silicon orglass, the surface must be activated prior to attaching the specificbinding partner. Also, polyelectrolyte interactions may be used toimmobilize a specific binding partner on a surface of a test piece byusing techniques and chemistries. The preferred method of attachment isby covalent means. Following attachment of a specific binding member,the surface may be further treated with materials such as serum,proteins, or other blocking agents to minimize non-specific binding. Thesurface also may be scanned either at the site of manufacture or pointof use to verify its suitability for assay purposes. The scanningprocess is not anticipated to alter the specific binding properties ofthe test piece.

[0209] While the present invention discloses the preference for the useof solid phases, it is contemplated that the reagents such asantibodies, proteins and peptides of the present invention can beutilized in non-solid phase assay systems. These assay systems are knownto those skilled in the art, and are considered to be within the scopeof the present invention.

[0210] It is contemplated that the reagent employed for the assay can beprovided in the form of a test kit with one or more containers such asvials or bottles, with each container containing a separate reagent suchas a probe, primer, monoclonal antibody or a cocktail of monoclonalantibodies, or a polypeptide (e.g. recombinantly, synthetically producedor purified) employed in the assay. The polypeptide is selected from thegroup consisting of SEQUENCE ID NO 24, SEQUENCE ID NO 25, SEQUENCE ID NO26, SEQUENCE ID NO 27, and fragments thereof. Other components such asbuffers, controls and the like, known to those of ordinary skill in art,may be included in such test kits. It also is contemplated to providetest kits which have means for collecting test samples comprisingaccessible body fluids, e.g., blood, urine, saliva and stool. Such toolsuseful for collection (“collection materials”) include lancets andabsorbent paper or cloth for collecting and stabilizing blood; swabs forcollecting and stabilizing saliva; cups for collecting and stabilizingurine or stool samples. Collection materials, papers, cloths, swabs,cups and the like, may optionally be treated to avoid denaturation orirreversible adsorption of the sample. The collection materials also maybe treated with or contain preservatives, stabilizers or antimicrobialagents to help maintain the integrity of the specimens. Test kitsdesigned for the collection, stabilization and preservation of testspecimens obtained by surgery or needle biopsy are also useful. It iscontemplated that all kits may be configured in two components which canbe provided separately; one component for collection and transport ofthe specimen and the other component for the analysis of the specimen.The collection component, for example, can be provided to the openmarket user while the components for analysis can be provided to otherssuch as laboratory personnel for determination of the presence, absenceor amount of analyte. Further, kits for the collection, stabilizationand preservation of test specimens may be configured for use byuntrained personnel and may be available in the open market for use athome with subsequent transportation to a laboratory for analysis of thetest sample.

[0211]E. coli bacteria (clone 2346388) has been deposited at theAmerican Type Culture Collection (A.T.C.C.), 12301 Parklawn Drive,Rockville, Md. 20852, as of ______, under the terms of the BudapestTreaty and will be maintained for a period of thirty (30) years from thedate of deposit, or for five (5) years after the last request for thedeposit, or for the enforceable period of the U.S. patent, whichever islonger. The deposit and any other deposited material described hereinare provided for convenience only, and are not required to practice thepresent invention in view of the teachings provided herein. The cDNAsequence in all of the deposited material is incorporated herein byreference. Clone 2346388 was accorded A.T.C.C. Deposit No. ______.

[0212] The present invention will now be described by way of examples,which are meant to illustrate, but not to limit, the scope of thepresent invention.

EXAMPLES Example 1 Identification of Prostate Tissue Library PS133Gene-specific Clones

[0213] A. Library Comparison of Expressed Sequence Tags (ESTs) orTranscript Images. Partial sequences of cDNA clone inserts, so-called“expressed sequence tags” (ESTs), were derived from cDNA libraries madefrom prostate tumor tissues, prostate non-tumor tissues and numerousother tissues, both tumor and non-tumor and entered into a database(LIFESEQ™ database, available from Incyte Pharmaceuticals, Palo Alto,Calif.) as gene transcript images. See International Publication No. WO95/20681. (A transcript image is a listing of the number of EST's foreach of the represented genes in a given tissue library. ESTs sharingregions of mutual sequence overlap are classified into clusters. Acluster is assigned a clone number from a representative 5′ EST. Often,a cluster of interest can be extended by comparing its consensussequence with sequences of other EST's which did not meet the criteriafor automated clustering. The alignment of all available clusters andsingle ESTs represent a contig from which a consensus sequence isderived.) The transcript images then were evaluated to identify ESTsequences that were representative primarily of the prostate tissuelibraries. These target clones then were ranked according to theirabundance (occurrence) in the target libraries and their absence frombackground libraries. Higher abundance clones with low backgroundoccurrence were given higher study priority. ESTs corresponding to theconsensus sequence of PS133 were found in 40.9% (9 of 22) of prostatetissue libraries. ESTs corresponding to the consensus sequence SEQUENCEID NO 7 (or fragments thereof) were found in only 2.3% (7 of 301) of theother, non-prostate, libraries of the data base. Therefore, theconsensus sequence or fragment thereof was found more than 17 times moreoften in prostate than non-prostate tissues. Overlapping clones 828846(SEQUENCE ID NO 1), 2346388 (SEQUENCE ID NO 2), 2531505 (SEQUENCE ID NO3),1725220 (SEQUENCE ID NO 4), 1856238 (SEQUENCE ID NO 5), and g1367041(SEQUENCE ID NO 6) were identified for further study. These representedthe minimum number of clones that were needed to form the contig andfrom which the consensus sequence provided herein (SEQUENCE ID NO 7) wasderived.

[0214] B. Generation of a Consensus Sequence. The nucleotide sequencesof clones 828846 (SEQUENCE ID NO 1), 2346388 (SEQUENCE ID NO 2), 2531505(SEQUENCE ID NO 3), 1725220 (SEQUENCE ID NO 4), 1856238 (SEQUENCE ID NO5), g1367041 (SEQUENCE ID NO 6) were entered in the Sequencher™ Program(available from Gene Codes Corporation, Ann Arbor, Mich., in order togenerate a nucleotide alignment (contig map) and then generate theirconsensus sequence (SEQUENCE ID NO 7). FIGS. 1A-1B show the nucleotidesequence alignment of these clones and their resultant nucleotideconsensus sequence (SEQUENCE ID NO 7). FIG. 2 presents the contig mapdepicting the clones SEQUENCE ID NOS 1-6 forming overlapping regions ofthe PS133 gene and the resultant consensus nucleotide sequence (SEQUENCEID NO 7) of these clones in a graphic display. Following this, athree-frame translation was performed on the consensus sequence(SEQUENCE ID NO 7). The first forward frame was found to have an openreading frame encoding a 250 residue amino acid sequence, which ispresented as SEQUENCE ID NO 24. The 250 residue polypeptide sequencedepicted in SEQUENCE ID NO 24 was compared with published sequencesusing software and techniques known to those skilled in the art. Thepolypeptide sequence of a murine serine protease termed “neuropsin” wasfound to be partially homologous to the PS133 polypeptide of SEQUENCE IDNO 24. The neuropsin enzyme is described by Z. Chen et al. J. Neurosci.15:5088-5097 (1995).

[0215] The consensus polypeptide of SEQUENCE ID NO 24 was also comparedagainst non-redundant protein data (non-redundant protein data is adatabase that has removed multiple references to proteins containing thesame sequence) from the GenBank and EMBL databases using a FASTAsoftware program. The comparison indicated significant homology withregions of human serine proteases. (Significant homology was determinedas a sequence of amino acid residues within the PS133 polypeptide whichshared identical or conserved amino acid residues to one of threecatalytically functional motifs within a serine proteinase, such thatone skilled in the art could identify the sequence as belonging to oneof the three serine protease motifs.) The regions were identified as thecatalytically functional motifs of serine protease (see Example 1C).

[0216] C. Alignment of PS133 Peptide with Known Serine Proteases

[0217] A detailed alignment of the PS133 polypeptide consensus sequence,SEQUENCE ID NO 24, with 47 known human serine proteases or serineprotease-like proteins (SEQUENCE ID NOS 28-74) was performed. The PS133consensus polypeptide was aligned against the primary sequence of thehuman serine proteases or serine protease-like domains from the GenBankdatabase (D. A. Benson et al., Nuc. Acids Res., Vol 25, pp 1-6, 1997,Oxford University Press) using the PILEUP computer program availablefrom Genetics Computer Group, Inc (University Research Park, Madison,Wis., USA). This alignment is shown in FIGS. 3A-D. The functional motifswhich are present in all catalytically functional serine proteases arealso present in the PS133 polypeptide. The functional motifs are markedwith an “*” in FIGS. 3A-D. Based on the homology of the PS133polypeptide with the functional motifs of other proteins in thedatabases, the program identified the PS133 polypeptide as a member ofthe human serine protease family.

Example 2 Sequencing of PS133 EST-specific Clones

[0218] The DNA sequence of clone 2346388 which comprises one of the5′-most ESTs of the PS133 gene contig was determined using dideoxytermination sequencing with dye terminators following known methods andis depicted as SEQUENCE ID NO 8. (F. Sanger et al., PNAS U.S.A. 74:5463(1977)).

[0219] Because the pSPORT1 vector (Life Technologies, Gaithersburg, Md.)contains universal priming sites just adjacent to the 3′ and 5′ ligationjunctions of the inserts, approximately 300 bases of the insert weresequenced in both directions using universal primers, SEQUENCE ID NO 11and SEQUENCE ID NO 12 (New England Biolabs, Beverly, Mass. and AppliedBiosystems Inc, Foster City, Calif., respectively). The sequencingreactions were run on a polyacrylamide denaturing gel, and the sequenceswere determined by an Applied Biosystems 377 Sequencer (available fromApplied Biosystems, Foster City, Calif.) or other sequencing apparatus.Additional sequencing primers, PS133. F1-5 and PS133. R1-6 (SEQUENCE IDNOS 13-17 and SEQUENCE ID NOS 18-23, respectively) were designed fromthe consensus sequence (SEQUENCE ID NO 7). These primers were used todetermine the remaining DNA sequence of the cloned insert from each DNAstrand, as previously described.

Example 3 Nucleic Acid Preparation

[0220] A. RNA Extraction from Tissue. Total RNA is isolated from solidprostate tissues or cells and from non-prostate tissues. Various methodsare utilized, including but not limited to the lithium chloride/ureatechnique, known and described in the art (Kato et al., J. Virol.61:2182-2191, [1987]), Ultraspec™ (Biotecx Laboratories, Inc., Houston,Tex.), and TRIzol™ (Life Technologies, Inc., Gaithersburg, Md.).

[0221] For northern blot analysis, the tissue is placed in a sterileconical tube on ice and 10-15 volumes of 3 M LiCl, 6 M urea, 5 mM EDTA,0.1 M β-mercaptoethanol, 50 mM Tris-HCl (pH 7.5) are added. The tissueis homogenized with a Polytron® homogenizer (Brinkman Instruments, Inc.,Westbury, N.Y.) for 30-50 sec on ice. The solution is transferred to a15 ml plastic centrifuge tube and placed overnight at −20° C. The tubeis centrifuged for 90 min at 9,000×g at 0-4° C., and the supernatant isimmediately decanted. Then, 10 ml of 3 M LiCl are added, the tube isvortexed for 5 sec and centrifuged for 45 min at 11,000×g at 0-4° C.Decanting, resuspension in LiCl, and centrifugation are repeated. Thefinal pellet is air dried and resuspended in 2 ml of 1 mM EDTA, 0.5%SDS, 10 mM Tris (pH 7.5). Then, 20 μl of Proteinase K (20 mg/ml) areadded, and the solution is incubated for 30 min at 37° C. withoccasional mixing. One-tenth volume (0.22-0.25 ml) of 3 M NaCl is added,and the solution is vortexed before transfer into another tube whichcontains 2 ml of phenol/chloroform/isoamyl alcohol (PCI). The tube isvortexed for 1-3 sec and centrifuged for 20 min at 3,000×g at 10° C. ThePCI extraction is repeated twice more, followed by two similarextractions with chloroform/isoamyl alcohol. The final aqueous solutionis transferred to a pre-chilled 15 ml corex glass tube containing 6 mlof 100% absolute ethanol, the tube is covered with parafilm and placedat −20° C. overnight. The tube is centrifuged for 30 min at 10,000×g at0-4° C., and the ethanol supernatant is decanted immediately. The RNApellet is washed four times with 10 ml of 75% ice-cold ethanol, followedeach time by centrifugation at 10,000×g for 10 min. The final pellet isair dried for 15 min at room temperature. The RNA is suspended in 0.5 mlof 10 mM Tris (pH 7.6), 1 mM EDTA, and its concentration is determinedspectrophotometrically. RNA samples are aliquoted and stored at −70° C.as ethanol precipitates.

[0222] The quality of the RNA is determined by agarose gelelectrophoresis (see Example 5) and staining with 0.5 μg/ml ethidiumbromide for one hour. RNA samples that do not contain intact 28S/18SrRNAs are excluded from the study.

[0223] Alternatively, for RT-PCR analysis, 1 ml of Ultraspec RNA reagentis added to 120 mg of pulverized tissue in a 2.0 ml polypropylenemicrofuge tube, homogenized with a Polytron® homogenizer (BrinkmanInstruments, Inc., Westbury, N.Y.) for 50 sec and left on ice for 5 min.Then, 0.2 ml of chloroform is added to each sample, followed byvortexing for 15 sec. The sample is left in ice for another 5 min,followed by centrifugation at 12,000×g for 15 min at 4° C. The upperlayer is collected and transferred to another RNase-free 2.0 mlmicrofuge tube. An equal volume of isopropanol is added to each sample,and the solution is placed on ice for 10 min. The sample is centrifugedat 12,000×g for 10 min at 4° C., and the supernatant is discarded. Theremaining pellet is washed twice with cold 75% ethanol, resuspended byvortexing, and the resuspended material is then re-pelleted bycentrifugation at 7500×g for 5 min at 4° C. Finally, the RNA pellet isdried in a speedvac for at least 5 min and reconstituted in RNase-freewater.

[0224] B. RNA Extraction from Blood Mononuclear Cells. Mononuclear cellsare isolated from blood samples from patients by centrifugation usingFicoll-Hypaque as follows. A 10 ml volume of whole blood is mixed withan equal volume of RPMI Medium (Life Technologies, Gaithersburg, Md.).This mixture is then underlayed with 10 ml of Ficoll-Hypaque (Pharmacia,Piscataway, N.J.) and centrifuged for 30 minutes at 200×g. The buffycoat containing the mononuclear cells is removed, diluted to 50 ml withDulbecco's PBS (Life Technologies, Gaithersburg, Md.) and the mixturecentrifuged for 10 minutes at 200×g. After two washes, the resultingpellet is resuspended in Dulbecco's PBS to a final volume of 1 ml.

[0225] RNA is prepared from the isolated mononuclear cells as describedby N. Kato et al., J. Virology 61: 2182-2191 (1987). Briefly, thepelleted mononuclear cells are brought to a final of 1 ml volume andthen are resuspended in 250 μL of PBS and mixed with 2.5 ml of 3M LiCl,6M urea, 5mM EDTA, 0.1M 2-mercaptoethanol, 50 mM Tris-HCl (pH 7.5). Theresulting mixture is homogenized and incubated at −20° C. overnight. Thehomogenate is spun at 8,000 RPM in a Beckman J2-21M rotor for 90 minutesat 0-4° C. The pellet is resuspended in 10 ml 3M LiCl by vortexing andthen spun at 10,000 RPM in a Beckman J2-21M rotor centrifuge for 45minutes at 0-4° C. The resuspending and pelleting steps then arerepeated. The pellet is resuspended in 2 ml of 1 mM EDTA, 0.5% SDS, 10mM Tris (pH 7.5) and 400 μg Proteinase K with vortexing and then it isincubated at 37° C. for 30 minutes with shaking. One tenth volume of 3MNaCl then is added and the vortexed mixture. Proteins are removed by twocycles of extraction with phenol/-chloroform/ isoamyl alcohol followedby one extraction with chloroform/ isoamyl alcohol. RNA is precipitatedby the addition of 6 ml of ethanol followed by overnight incubation at−20° C. After the precipitated RNA is collected by centrifugation, thepellet is washed 4 times in 75% ethanol. The pelleted RNA is thendissolved in 1 mM EDTA, 10 mM Tris-HCl (pH 7.5).

[0226] Non-prostate tissues are used as negative controls. The mRNA canbe further purified from total RNA by using commercially available kitssuch as oligo dT cellulose spin columns (RediCol™ from Pharmacia,Uppsala, Sweden) for the isolation of poly-adenylated RNA. Total or mRNAcan be dissolved in lysis buffer (5M guanidine thiocyanate, 0.1M EDTA,pH 7.0) for analysis in the ribonuclease protection assay.

[0227] C. RNA Extraction from polysomes. Tissue is minced in saline at4° C. and mixed with 2.5 volumes of 0.8 M sucrose in a TK₁₅₀M (150 mMKCl, 5 mM MgCl₂,50 mM Tris-HCl, pH 7.4) solution containing 6 mM2-mercaptoethanol. The tissue is homogenized in a Teflon-glass Potterhomogenizer with five strokes at 100-200 rpm followed by six strokes ina Dounce homogenizer, as described by B. Mechler, Methods in Enzymology152:241-248 (1987). The homogenate then is centrifuged at 12,000×g for15 min at 4° C. to sediment the nuclei. The polysomes are isolated bymixing 2 ml of the supernatant with 6 ml of 2.5 M sucrose in TK₁₅₀M andlayering this mixture over 4 ml of 2.5 M sucrose in TK₁₅₀M in a 38 mlpolyallomer tube. Two additional sucrose TK₁₅₀M solutions aresuccessively layered onto the extract fraction; a first layer of 13 ml2.05 M sucrose followed by a second layer of 6 ml of 1.3 M sucrose. Thepolysomes are isolated by centrifuging the gradient at 90,000×g for 5 hat 4° C. The fraction then is taken from the 1.3 M sucrose/2.05 Msucrose interface with a siliconized pasteur pipette and diluted in anequal volume of TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA). An equal volumeof 90° C. SDS buffer (1% SDS, 200 mM NaCl, 20 mM Tris-HCl, pH 7.4) isadded and the solution is incubated in a boiling water bath for 2 min.Proteins next are digested with a Proteinase-K digestion (50 mg/ml) for15 min at 37° C. The mRNA is purified with 3 equal volumes ofphenol-chloroform extractions followed by precipitation with 0.1 volumeof 2 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol at −20° C.overnight. The precipitated RNA is recovered by centrifugation at12,000×g for 10 min at 4° C. The RNA is dried and resuspended in TE (pH7.4) or distilled water. The resuspended RNA then can be used in a slotblot or dot blot hybridization assay to check for the presence of PS133mRNA (see Example 6).

[0228] The quality of nucleic acid and proteins is dependent on themethod of preparation used. Each sample may require a differentpreparation technique to maximize isolation efficiency of the targetmolecule. These preparation techniques are within the skill of theordinary artisan.

Example 4 Ribonuclease Protection Assay

[0229] A. Synthesis of Labeled Complementary RNA (cRNA) HybridizationProbe and Unlabeled Sense Strand. Labeled antisense and unlabeled senseriboprobes are transcribed from the PS133 gene cDNA sequence whichcontains a 5′ RNA polymerase promoter such as SP6 or T7. The sequencemay be from a vector containing the appropriate PS133 cDNA insert, orfrom a PCR-generated product of the insert using PCR primers whichincorporate a 5′ RNA polymerase-promoter sequence. For example, thedescribed plasmid, clone 2346388 or other comparable clone, containingthe PS133 gene cDNA sequence, flanked by opposed SP6 and T7 polymerasepromoters, is purified using Qiagen Plasmid Purification Kit (Qiagen,Chatsworth, Calif.). Then 10 μg of the plasmid are linearized by cuttingwith 10 U DdeI restriction enzyme for 1 h at 37° C. The linearizedplasmid is purified using QIAprep kits (Qiagen, Chatsworth, Calif.) andused for the synthesis of antisense transcript from the appropriate SP6or T7 promoter using the Riboprobe® in vitro Transcription System(Promega Corporation, Madison, Wis.), as described by the supplier'sinstructions, incorporating either 6.3 μM (alpha³²P) UTP (Amersham LifeSciences, Inc. Arlington Heights, Ill.) or 100-500 μM biotinylated UTPas a label. To generate the sense strand, 10 μg of the purified plasmidare cut with restriction enzymes 10U XbaI and 10 U NotI, and transcribedas above from the appropriate SP6 or T7 promoter. Both sense andantisense strands are isolated by spin column chromatography. Unlabeledsense strand is quantitated by UV absorption at 260 nm.

[0230] B. Hybridization of Labeled Probe to Target. Frozen tissue ispulverized to powder under liquid nitrogen and 100-500 mg are dissolvedin 1 ml of lysis buffer, available as a component of the Direct Protect™Lysate RNase Protection kit (Ambion, Inc., Austin, Tex.). Furtherdissolution can be achieved using a tissue homogenizer. In addition, adilution series of a known amount of sense strand in mouse liver lysateis made for use as a positive control. Finally, 45 μl of solubilizedtissue or diluted sense strand is mixed directly with either 1) 1×10⁵cpm of radioactively labeled probe or 2) 250 pg of non-isotopicallylabeled probe in 5 μl of lysis buffer. Hybridization is allowed toproceed overnight at 37° C. See, T. Kaabache et al., Anal. Biochem.232:225-230 (1995).

[0231] C. RNase Digestion. RNA that is not hybridized to probe isremoved from the reaction as per the Direct Protect™ protocol using asolution of RNase A and RNase T1 for 30 min at 37° C., followed byremoval of RNase by Proteinase-K digestion in the presence of sodiumsarcosyl. Hybridized fragments protected from digestion are thenprecipitated by the addition of an equal volume of isopropanol andplaced at −70° C. for 3 h. The precipitates are collected bycentrifugation at 12,000×g for 20 min.

[0232] D. Fragment Analysis. The precipitates are dissolved indenaturing gel loading dye (80% formamide, 10 mM EDTA (pH 8.0), 1 mg/mlxylene cyanol, 1 mg/ml bromophenol blue), heat denatured, andelectrophoresed in 6% polyacrylamide TBE, 8 M urea denaturing gels. Thegels are imaged and analyzed using the STORM™ storage phosphorautoradiography system (Molecular Dynamics, Sunnyvale, Calif.).Quantitation of protected fragment bands, expressed in femtograms (fg),is achieved by comparing the peak areas obtained from the test samplesto those from the known dilutions of the positive control sense strand(see Section B, supra). The results are expressed in molecules of PS133RNA/cell and as a image rating score. In cases where non-isotopic labelsare used, hybrids are transferred from the gels to membranes (nylon ornitrocellulose) by blotting and then analyzed using detection systemsthat employ streptavidin alkaline phosphatase conjugates andchemiluminesence or chemifluoresence reagents. High level expression ofmRNA corresponding to a sequence selected from the group consisting ofSEQUENCE ID NOS 1-8, and fragments or complements thereof, indicate thepresence of PS133 mRNA(s), suggesting a diagnosis of a prostate tissuedisease or condition, such as prostate cancer.

Example 5 Northern Blotting

[0233] The northern blot technique is used to identify a specific sizeRNA fragment from a complex population of RNA using gel electrophoresisand nucleic acid hybridization. Northern blotting is well-knowntechnique in the art. Briefly, 5-10 μg of total RNA (see Example 3) areincubated in 15 μl of a solution containing 40 mMmorphilinopropanesulfonic acid (MOPS) (pH 7.0), 10 mM sodium acetate, 1mM EDTA, 2.2 M formaldehyde, 50% v/v formamide for 15 min at 65° C. Thedenatured RNA is mixed with 2 μl of loading buffer (50% glycerol, 1 mMEDTA, 0.4% bromophenol blue, 0.4% xylene cyanol) and loaded into adenaturing 1.0% agarose gel containing 40 mM MOPS (pH 7.0), 10 mM sodiumacetate, 1 mM EDTA and 2.2 M formaldehyde. The gel is electrophoresed at60 V for 1.5 h and rinsed in RNAse free water. RNA is transferred fromthe gel onto nylon membranes (Brightstar-Plus, Ambion, Inc., Austin,Tex.) for 1.5 hours using the downward alkaline capillary transfermethod (Chomczynski, Anal. Biochem. 201:134-139, 1992). The filter isrinsed with 1×SSC, and RNA is crosslinked to the filter using aStratalinker (Stratagene, Inc., La Jolla, Calif.) on theautocrosslinking mode and dried for 15 min. The membrane is then placedinto a hybridization tube containing 20 ml of preheated prehybridizationsolution (5×SSC, 50% formamide, 5× Denhardt's solution, 100 μg/mldenatured salmon sperm DNA) and incubated in a 42° C. hybridization ovenfor at least 3 hr. While the blot is prehybridizing, a ³²P -labeledrandom-primed probe is generated using the PS133 insert fragment(obtained by digesting clone 2346388 or another comparable clone, withXbaI and NotI) using Random Primer DNA Labeling System (LifeTechnologies, Inc., Gaithersburg, Md.) according to the manufacturer'sinstructions. Half of the probe is boiled for 10 min, quick chilled onice and added to the hybridization tube. Hybridization is carried out at42° C. for at least 12 hr. The hybridization solution is discarded andthe filter is washed in 30 ml of 3×SSC, 0.1% SDS at 42° C. for 15 min,followed by 30 ml of 3×SSC, 0.1% SDS at 42° C. for 15 min. The filter iswrapped in saran wrap, exposed to Kodak XAR-Omat film for 8-96 hr, andthe film is developed for analysis. High level of expression of mRNAcorresponding to a sequence selected from the group consisting ofSEQUENCE ID NOS 1-8, and fragments or complements thereof, is anindication of the presence of PS133 mRNA, suggesting a diagnosis of aprostate tissue disease or condition, such as prostate cancer.

Example 6 Dot Blot/Slot Blot

[0234] Dot and slot blot assays are quick methods to evaluate thepresence of a specific nucleic acid sequence in a complex mix of nucleicacid. To perform such assays, up to 50 μg of RNA is mixed in 50 μl of50% formamide, 7% formaldehyde, 1×SSC, incubated 15 min at 68° C., andthen cooled on ice. Then, 100 μl of 20×SSC is added to the RNA mixtureand loaded under vacuum onto a manifold apparatus that has a preparednitrocellulose or nylon membrane. The membrane is soaked in water, 20×SSC for 1 hour, placed on two sheets of 20×SSC prewet Whatman #3 filterpaper, and loaded into a slot blot or dot blot vacuum manifoldapparatus. The slot blot is analyzed with probes prepared and labeled asdescribed in Example 4, supra. Detection of mRNA corresponding to asequence selected from the group consisting of SEQUENCE ID NOS 1-8, andfragments or complements thereof, is an indication of the presence ofPS133, suggesting a diagnosis of a prostate tissue disease or condition,such as prostate cancer.

[0235] Other methods and buffers which can be utilized in the methodsdescribed in Examples 5 and 6, but not specifically detailed herein, areknown in the art and are described in J. Sambrook et al., supra which isincorporated herein by reference.

Example 7 In Situ Hybridization

[0236] This method is useful to directly detect specific target nucleicacid sequences in cells using detectable nucleic acid hybridizationprobes.

[0237] Tissues are prepared with cross-linking fixative agents such asparaformaldehyde or glutaraldehyde for maximum cellular RNA retention.See, L. Angerer et al., Methods in Cell Biol. 35:37-71 (1991). Briefly,the tissue is placed in greater than 5 volumes of 1% glutaraldehyde in50 mM sodium phosphate, pH 7.5 at 4° C. for 30 min. The solution ischanged with fresh glutaraldehyde solution (1% glutaraldehyde in 50 mMsodium phosphate, pH 7.5) for a further 30 min fixing. The fixingsolution should have an osmolality of approximately 0.375% NaCl. Thetissue is washed once in isotonic NaCl to remove the phosphate.

[0238] The fixed tissues then are embedded in paraffin as follows. Thetissue is dehydrated though a series of ethanol concentrations for 15min each: 50% (twice), 70% (twice), 85%, 90% and then 100% (twice).Next, the tissue is soaked in two changes of xylene for 20 min each atroom temperature. The tissue is then soaked in two changes of a 1:1mixture of xylene and paraffin for 20 min each at 60° C.; and then inthree final changes of paraffin for 15 min each.

[0239] Next, the tissue is cut in 5 μm sections using a standardmicrotome and placed on a slide previously treated with a tissueadhesive such as 3-aminopropyltriethoxysilane.

[0240] Paraffin is removed from the tissue by two 10 min xylene soaksand rehydrated in a series of ethanol concentrations: 99% twice, 95%,85%, 70%, 50%, 30%, and then distilled water twice. The sections arepre-treated with 0.2 M HCl for 10 min and permeabilized with 2 μg/mlProteinase-K at 37° C. for 15 min.

[0241] Labeled riboprobes transcribed from the PS133 gene plasmid (seeExample 4) are hybridized to the prepared tissue sections and incubatedovernight at 56° C. in 3×standard saline extract and 50% formamide.Excess probe is removed by washing in 2×standard saline citrate and 50%formamide followed by digestion with 100 μg/ml RNase A at 37° C. for 30min. Fluorescence probe is visualized by illumination with ultraviolet(UV) light under a microscope. Fluorescence in the cytoplasm isindicative of PS133 mRNA. Alternatively, the sections can be visualizedby autoradiography.

Example 8 Reverse Transcription PCR

[0242] A. One Step RT-PCR Assay. Target-specific primers are designed todetect the above-described target sequences by reverse transcription PCRusing methods known in the art. One step RT-PCR is a sequentialprocedure that performs both RT and PCR in a single reaction mixture.The procedure is performed in a 200 μl reaction mixture containing 50 mM(N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15, 81.7 mM KOAc, 33.33 mM KOH,0.01 mg/ml bovine serum albumin, 0.1 mM ethylene diaminetetraaceticacid, 0.02 mg/ml NaN_(3,8)% w/v glycerol, 150 μM each of dNTP, 0.25 μMeach primer, 5U rTth polymerase, 3.25 mM Mn(OAc)₂ and 5 μl of target RNA(see Example 3). Since RNA and the rTth polymerase enzyme are unstablein the presence of Mn(OAc)₂, the Mn(OAc)₂ should be added just beforetarget addition. Optimal conditions for cDNA synthesis and thermalcycling readily can be determined by those skilled in the art. Thereaction is incubated in a Perkin-Elmer Thermal Cycler 480. Optimalconditions for cDNA synthesis and thermal cycling can readily bedetermined by those skilled in the art. Conditions which may be founduseful include cDNA synthesis at 60°-70° C. for 15-45 min and 30-45amplification cycles at 94° C., 1 min; 55°-70° C., 1 min; 72° C., 2 min.One step RT-PCR also may be performed by using a dual enzyme procedurewith Taq polymerase and a reverse transcriptase enzyme, such as MMLV orAMV RT enzymes.

[0243] B. Traditional RT-PCR. Alternatively, a traditional two-stepRT-PCR reaction may be performed, as described by K. Q. Hu et al.,Virology 181:721-726 (1991), as follows. The extracted mRNA istranscribed in a 25 μl reaction mixture containing 10 mM Tris-HCl, pH8.3, 5 MM MgCl₂, 500 μM dNTP, 20 U RNasin, 1 μM antisense primer and 25U AMV (avian myeloblastosis virus) or MMLV (Moloney murine leukemiavirus) reverse transcriptase. Reverse transcription is performed at37-45° C. for 30-60 min, followed by further incubation at 95° C. for 5min to inactivate the RT. PCR is performed using 10 μl of the cDNAreaction in a final PCR reaction volume of 50 μl containing 10 mMTris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 200 μM dNTP, 0.5 μM of eachprimer and 2.5 U of Taq polymerase. Optimal conditions for cDNAsynthesis and thermal cycling can be readily determined by those skilledin the art. The reaction is incubated in a Perkin-Elmer Thermal Cycler480 or other comparable instrument. Conditions which may be found usefulinclude 30-45 cycles of amplification (94° C., 1 min; 55-70° C., 1 min;72° C., 2 min), final extension (72° C., 10 min) and soak at 4° C.

[0244] C. PCR Fragment Analysis. The correct products then can beverified by size determination using gel electrophoresis withfluorescent intercalators, such as SYBR™ Green I (Molecular Probes,Eugene, Oreg.) and imaged using a STORM imaging system, or also verifiedby Southern, dot or slot blot analysis using a labeled probe against theinternal sequences of the PCR product. The probes also may bepolynucleotides analogs, such as morpholinos or peptide nucleic acidsanalogs (PNAs). Detection of a product comprising a sequence selectedfrom the group consisting of SEQUENCE ID NOS 1-8, and fragments orcomplements thereof, is indicative of the presence of PS133 mRNA(s),suggesting a diagnosis of a prostate tissue disease or condition, suchas prostate cancer.

Example 9 OH-PCR

[0245] A. Probe selection and Labeling. Target-specific primers andprobes are designed to detect the above-described target sequences byoligonucleotide hybridization PCR. International Publication Nos WO92/10505, published 25 Jun. 1992, and WO 92/11388, published 9 Jul.1992, teach methods for labeling oligonucleotides at their 5′ and 3′ends, respectively. According to one known method for labeling anoligonucleotide, a label-phosphoramidite reagent is prepared and used toadd the label to the oligonucleotide during its synthesis. For example,see N. T. Thuong et al., Tet. Letters 29(46):5905-5908 (1988); or J. S.Cohen et al., published U.S. patent application Ser. No. 07/246,688(NTIS ORDER No. PAT-APPL-7-246,688) (1989). Preferably, probes arelabeled at their 3′ end to prevent participation in PCR and theformation of undesired extension products. For one step OH-PCR, theprobe should have a T_(M) at least 15° C. below the T_(M) of theprimers. The primers and probes are utilized as specific bindingmembers, with or without detectable labels, using standardphosphoramidite chemistry and/or post-synthetic labeling methods whichare well-known to one skilled in the art.

[0246] B. One Step Oligo Hybridization PCR. OH-PCR is performed on a 200μl reaction containing 50 mM (N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15,81.7 mM KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1 mMethylene diaminetetraacetic acid, 0.02 mg/ml NaN₃, 8% w/v glycerol, 150μM each of dNTP, 0.25 μM each primer, 3.75 nM probe, 5U rTth polymerase,3.25 mM Mn(OAc)₂ and 5 μl blood equivalents of target (see Example 3).Since RNA and the rTth polymerase enzyme are unstable in the presence ofMn(OAc)₂, the Mn(OAc)₂ should be added just before target addition. Thereaction is incubated in a Perkin-Elmer Thermal Cycler 480. Optimalconditions for cDNA synthesis and thermal cycling can be readilydetermined by those skilled in the art. Conditions which may be founduseful include cDNA synthesis (60° C., 30 min), 30-45 amplificationcycles (94° C., 40 sec; 55-70° C., 60 sec), oligo-hybridization (97° C.,5 min; 15° C., 5 min; 15° C. soak). The correct reaction productcontains at least one of the strands of the PCR product and aninternally hybridized probe.

[0247] C. OH-PCR Product Analysis. Amplified reaction products aredetected on an LCx® analyzer system (available from Abbott Laboratories,Abbott Park, Ill.). Briefly, the correct reaction product is captured byan antibody labeled microparticle at a capturable site on either the PCRproduct strand or the hybridization probe, and the complex is detectedby binding of a detectable antibody conjugate to either a detectablesite on the probe or the PCR strand. Only a complex containing a PCRstrand hybridized with the internal probe is detectable. The detectionof this complex then is indicative of the presence of PS133 mRNA,suggesting a diagnosis of a prostate disease or condition, such asprostate cancer.

[0248] Many other detection formats exist which can be used and/ormodified by those skilled in the art to detect the presence of amplifiedor non-amplified PS133-derived nucleic acid sequences including, but notlimited to, ligase chain reaction (LCR, Abbott Laboratories, AbbottPark, Ill.); Q-beta replicase (Gene-Trak™, Naperville, Ill.), branchedchain reaction (Chiron, Emeryville, Calif.) and strand displacementassays (Becton Dickinson, Research Triangle Park, N.C.).

Example 10 Synthetic Peptide Production

[0249] Synthetic peptides are modeled and then prepared based upon thepredicted amino acid sequence of the PS133 polypeptide consensussequence (see example 1). Peptides modeled for PS133 include SEQUENCE IDNO 25, SEQUENCE ID NO 26, SEQUENCE ID NO 27, and fragments thereofderived from SEQUENCE ID NO 24. All peptides are synthesized on aSymphony Peptide Synthesizer (available from Rainin Instrument Co,Emeryville Calif.) or similar instrument, using FMOC chemistry, standardcycles and in-situ HBTU activation. Cleavage and deprotection conditionsare as follows: a volume of 2.5 ml of cleavage reagent (77.5% v/vtrifluoroacetic acid, 15% v/v ethanedithiol, 2.5% v/v water, 5% v/vthioanisole, 1-2% w/v phenol) is added to the resin, and agitated atroom temperature for 2-4 hours. Then the filtrate is removed and thepeptide is precipitated from the cleavage reagent with cold diethylether. Each peptide is filtered, purified via reverse-phase preparativeHPLC using a water/acetonitrile/0.1% TFA gradient, and lyophilized. Theproduct is confirmed by mass spectrometry (see Example 12).

[0250] Disulfide bond formation is accomplished using auto-oxidationconditions, as follows: the peptide is dissolved in a minimum amount ofDMSO (approximately 10 ml) before adding buffer (0.1 M Tris-HCl, pH 6.2)to a concentration of 0.3-0.8 mg/ml. The reaction is monitored by HPLCuntil complete formation of the disulfide bond, followed byreverse-phase preparative HPLC using a water/acetonitrile/0.1% TFAgradient and lyophilization. The product then is confirmed by massspectrometry (see Example 12).

[0251] The purified peptides can be conjugated to Keyhole LimpetHemocyanin or other immunoreactive molecule with glutaraldehyde, mixedwith adjuvant, and injected into animals.

Example 11a Expression of Protein in a Cell Line Using Plasmid 577

[0252] A. Construction of a PS133 Expression Plasmid. Plasmid 577,described in U.S. patent application Ser. No. 08/478,073, filed Jun. 7,1995 and incorporated herein by reference, has been constructed for theexpression of secreted antigens in a permanent cell line. This plasmidcontains the following DNA segments: (a) a 2.3 Kb fragment of pBR322containing bacterial beta-lactamase and origin of DNA replication; (b) a1.8 Kb cassette directing expression of a neomycin resistance gene undercontrol of HSV-1 thymidine kinase promoter and poly-A addition signals;(c) a 1.9 Kb cassette directing expression of a dihydrofolate reductasegene under the control of an SV-40 promoter and poly-A addition signals;(d) a 3.5 Kb cassette directing expression of a rabbit immunoglobulinheavy chain signal sequence fused to a modified hepatitis C virus (HCV)E2 protein under the control of the Simian Virus 40 T-Ag promoter andtranscription enhancer, the hepatitis B virus surface antigen (HBsAg)enhancer I followed by a fragment of Herpes Simplex Virus-1 (HSV-1)genome providing poly-A addition signals; and (e) a residual 0.7 Kbfragment of Simian Virus 40 genome late region of no function in thisplasmid. All of the segments of the vector were assembled by standardmethods known to those skilled in the art of molecular biology.

[0253] Plasmids for the expression of secretable PS133 proteins areconstructed by replacing the hepatitis C virus E2 protein codingsequence in plasmid 577 with that of a PS133 polynucleotide sequenceselected from the group consisting of SEQUENCE ID NOS 1-8, and fragmentsor complements thereof, as follows. Digestion of plasmid 577 with XbaIreleases the hepatitis C virus E2 gene fragment. The resulting plasmidbackbone allows insertion of the PS133 cDNA insert downstream of therabbit immunoglobulin heavy chain signal sequence which directs theexpressed proteins into the secretory pathway of the cell. The PS133cDNA fragment is generated by PCR using standard procedures. Encoded inthe sense PCR primer sequence is an XbaI site, immediately followed by a12 nucleotide sequence that encodes the amino acid sequenceSer-Asn-Glu-Leu (“SNEL”) to promote signal protease processing,efficient secretion and final product stability in culture fluids.Immediately following this 12 nucleotide sequence the primer containsnucleotides complementary to template sequences encoding amino acids ofthe PS133 gene. The antisense primer incorporates a sequence encodingthe following eight amino acids just before the stop codons:Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQUENCE ID NO 75). Within thissequence is incorporated a recognition site to aid in analysis andpurification of the PS133 protein product. A recognition site (termed“FLAG”) that is recognized by a commercially available monoclonalantibody designated anti-FLAG M2 (Eastman Kodak, Co., New Haven, Conn.)can be utilized, as well as other comparable sequences and theircorresponding antibodies. For example, PCR is performed using GeneAmp®reagents obtained from Perkin-Elmer-Cetus, as directed by the supplier'sinstructions. PCR primers are used at a final concentration of 0.5 μM.PCR is performed on the PS133 plasmid template in a 100 μl reaction for35 cycles (94° C., 30 seconds; 55° C., 30 seconds; 72° C., 90 seconds)followed by an extension cycle of 72° C. for 10 min.

[0254] B. Transfection of Dihydrofolate Reductase Deficient ChineseHamster Ovary Cells. The plasmid described supra is transfected intoCHO/dhfr− cells (DXB-111, Uriacio et al., PNAS 77:4451-4466 (1980)).These cells are available from the A.T.C.C., 12301 Parklawn Drive,Rockville, Md. 20852, under Accession No. CRL 9096. Transfection iscarried out using the cationic liposome-mediated procedure described byP. L. Felgner et al., PNAS 84:7413-7417 (1987). Particularly, CHO/dhfr-cells are cultured in Ham's F-12 media supplemented with 10% fetal calfserum, L-glutamine (1 mM) and freshly seeded into a flask at a densityof 5-8×10⁵ cells per flask. The cells are grown to a confluency ofbetween 60 and 80% for transfection. Twenty micrograms (20, g) ofplasmid DNA is added to 1.5 ml of Opti-MEM I medium and 100 μl ofLipofectin Reagent (Gibco-BRL; Grand Island, N.Y.) are added to a second1.5 ml portion of Opti-MEM I media. The two solutions are mixed andincubated at room temperature for 20 min. After the culture medium isremoved from the cells, the cells are rinsed 3 times with 5 ml ofOpti-MEM I medium. The Opti-MEM I-Lipofection-plasmid DNA solution thenis overlaid onto the cells. The cells are incubated for 3 h at 37° C.,after which time the Opti-MEM I-Lipofectin-DNA solution is replaced withculture medium for an additional 24 h prior to selection.

[0255] C. Selection and Amplification. One day after transfection, cellsare passaged 1:3 and incubated with dhfr/G418 selection medium(hereafter, “F-12 minus medium G”). Selection medium is Ham's F-12 withL-glutamine and without hypoxanthine, thymidine and glycine (JRHBiosciences, Lenexa, Kans.) and 300 μg per ml G418 (Gibco-BRL; GrandIsland, N.Y.). Media volume-to-surface area ratios of 5 ml per 25 cm²are maintained. After approximately two weeks, DHFR/G418 cells areexpanded to allow passage and continuous maintenance in F-12 minusmedium G.

[0256] Amplification of each of the transfected PS133 cDNA sequences isachieved by stepwise selection of DHFR⁺, G418⁺ cells with methotrexate(reviewed by R. Schimke, Cell 37:705-713 [1984]). Cells are incubatedwith F-12 minus medium G containing 150 nM methotrexate (MTX) (Sigma,St. Louis, Mo.) for approximately two weeks until resistant coloniesappear. Further gene amplification is achieved by selection of 150 nMadapted cells with 5 μM MTX.

[0257] D. Antigen Production. F-12 minus medium G supplemented with 5 μMMTX is overlaid onto just confluent monolayers for 12 to 24 h at 37° C.in 5% CO₂. The growth medium is removed and the cells are rinsed 3 timeswith Dulbecco's phosphate buffered saline (PBS) (with calcium andmagnesium) (Gibco-BRL; Grand Island, N.Y.) to remove the remainingmedia/serum which may be present. Cells then are incubated with VAScustom medium (VAS custom formulation with L-glutamine with HEPESwithout phenol red, available from JRH Bioscience; Lenexa, KS, productnumber 52-08678P), for 1 h at 37° C. in 5% CO₂. Cells then are overlaidwith VAS for production at 5 ml per T flask. Medium is removed afterseven days of incubation, retained, and then frozen to awaitpurification with harvests 2, 3 and 4. The monolayers are overlaid withVAS for 3 more seven day harvests.

[0258] E. Analysis of Prostate Tissue Gene PS133 Antigen Expression.Aliquots of VAS supernatants from the cells expressing the PS133 proteinconstruct are analyzed, either by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) using standard methods and reagents known in the art (Laemmlidiscontinuous gels), or by mass spectrometry.

[0259] F. Purification. Purification of the PS133 protein containing theFLAG sequence is performed by immunoaffinity chromatography using anaffinity matrix comprising anti-FLAG M2 monoclonal antibody covalentlyattached to agarose by hydrazide linkage (Eastman Kodak Co., New Haven,Conn.). Prior to affinity purification, protein in pooled VAS mediumharvests from roller bottles is exchanged into 50 mM Tris-HCl (pH 7.5),150 mM NaCl buffer using a Sephadex G-25 (Pharmacia Biotech Inc.,Uppsala, Sweden) column. Protein in this buffer is applied to theanti-FLAG M2 antibody affinity column. Non-binding protein is eluted bywashing the column with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl buffer.Bound protein is eluted using an excess of FLAG peptide in 50 mMTris-HCl (pH 7.5), 150 mM NaCl. The excess FLAG peptide can be removedfrom the purified PS133 protein by gel electrophoresis or HPLC.

[0260] Although plasmid 577 is utilized in this example, it is known tothose skilled in the art that other comparable expression systems, suchas CMV, can be utilized herein with appropriate modifications in reagentand/or techniques and are within the skill of the ordinary artisan.

[0261] The largest cloned insert containing the coding region of thePS133 gene is then sub-cloned into either (i) a eukaryotic expressionvector which may contain, for example, a cytomegalovirus (CMV) promoterand/or protein fusible sequences which aid in protein expression anddetection, or (ii) a bacterial expression vector containing asuperoxide-dismutase (SOD) and CMP-KDO synthetase (CKS) or other proteinfusion gene for expression of the protein sequence. Methods and vectorswhich are useful for the production of polypeptides which contain fusionsequences of SOD are described in EPO 0196056, published Oct. 1, 1986,which is incorporated herein by reference and those containing fusionsequences of CKS are described in EPO Publication No. 0331961, publishedSep. 13, 1989, which publication is also incorporated herein byreference. This so-purified protein can be used in a variety oftechniques, including but not limited to animal immunization studies,solid phase immunoassays, etc.

Example 11b Expression of Protein in a Cell Line Using pcDNA3.1/Myc-His

[0262] A. Construction of a PS133 Expression Plasmid. PlasmidpcDNA3.1/Myc-His (Cat.# V855-20, Invitrogen, Carlsbad, Calif.) has beenconstructed, in the past, for the expression of secreted antigens bymost mammalian cell lines. Expressed protein inserts are fused to amyc-his peptide tag. The myc-his tag is a 21 residue amino acid sequencehaving the following sequence:Glu-Gln-Lys-Leu-Ee-Ser-Glu-Glu-Asp-Leu-Asn-Met-His-Thr-Glu-His-His-His-His-His-His(SEQUENCE ID NO 76) and comprises a myc epitope and a polyhistidinesequence which are useful for the purification of an expressed fusionprotein using either anti-myc or anti-his affinity columns, ormetalloprotein binding columns.

[0263] Plasmids for the expression of secretable PS133 proteins areconstructed by inserting a PS133 polynucleotide sequence selected fromthe group consisting of SEQUENCE ID NOS 1-8, and fragments orcomplements thereof. Prior to construction of a PS133 expressionplasmid, the PS133 cDNA sequence is first cloned into a pCR®-Bluntvector as follows.

[0264] The PS133 cDNA fragment is generated by PCR using standardprocedures. For example, PCR is performed using Stratagene® reagentsobtained from Stratagene, La Jolla, Calif., as directed by thesupplier's instructions. PCR primers are used at a final concentrationof 0.5 μM. PCR using 5 U of pfu polymerase (Stratagene) is performed onthe PS133 plasmid template (see Example 2) in a 50 μl reaction for 30cycles (94° C., 1 min; 65° C., 1.5 min; 72° C., 3 min) followed by anextension cycle at 72° C. for 8 min. The sense PCR primer sequencecomprises nucleotides which are either complementary to the pINCY vectordirectly upstream of the PS133 gene insert or which incorporate a 5′EcoRI restriction site, an adjacent downstream protein translationconsensus initiator, and a 3′ nucleic acid sequence which is the samesense as the 5′-most end of the PS133 cDNA insert. The antisense primerincorporates a 5′ NotI restriction sequence and a sequence complementaryto the 3′ end of the PS133 cDNA insert just upstream of the 3′-most,in-frame stop codon. Five microliters (5 μl) of the resultingblunt-ended PCR product are ligated into 25 ng of linearized pCR®-Bluntvector (Invitrogen, Carlsbad, Calif.) interrupting the lethal ccdB geneof the vector. The resulting ligated vector is transfected into TOP10 E.coli (Invitrogen , Carlsbad, Calif.) using a One Shot™ transformationkit (Invitrogen, Carlsbad, Calif.) following the supplier's directions.The transfected cells are grown on LB-Kan (50 μg/ml kanamycin) selectionplates at 37° C. Only cells containing a plasmid with an interruptedccdB gene will grow after transfection (Grant, S. G. N., PNAS USA87:4645-4649 (1990)). Transfected colonies are picked and grown up in 3ml of LB-Kan broth at 37° C. Plasmid DNA is isolated using a QIAprep®(Qiagen Inc., Santa Clarita, Calif.) procedure, as directed by thesupplier's instructions. The DNA is cut with EcoRI or SnaBI, and NotIrestriction enzymes to release the PS133 insert fragment. The fragmentis run on 1% Seakem® LE agarose/0.5 μg/ml ethidium bromide/TE gel,visualized by UV irradiation, excised and purified using QIAquick™(Qiagen Inc., Santa Clarita, Calif.) procedures, as directed by thesupplier's instructions.

[0265] The pcDNA3.1/Myc-His plasmid DNA is linearized by digestion withEcoRI or SnaBI, and NotI in the polylinker region of the plasmid DNA.The resulting plasmid DNA backbone allows insertion of the PS133purified cDNA fragment, supra, downstream of a CMV promoter whichdirects expression of the proteins in mammalian cells. The ligatedplasmid is transfected into DH5 alpha™ cells (GibcoBRL, Gaithersburg,Md.) as directed by the supplier's instructions. Briefly, 10 ng ofpcDNA3.1/Myc-His containing a PS133 insert is added to 50 μl ofcompetent DH5 alpha cells, and the contents are mixed gently. Themixture is incubated on ice for 30 min, heat shocked for 20 sec at 37°C., and placed on ice for an additional 2 min. Upon addition of 0.95 mlof LB medium, the mixture is incubated for 1 h at 37° C. while shakingat 225 rpm. The transfected cells are then plated onto 100 mm LB/Amp (50μg/ml ampicillin) plates and grown at 37° C. Colonies are picked andgrown in 3 ml of LB/Amp broth. Plasmid DNA is purified using a QIAprep®kit. Presence of the insert is confirmed using techniques known to thoseskilled in the art including, but not limited to, restriction digestionand gel analysis. See, e.g., J. Sambrook et al., supra.

[0266] B. Transfection of Human Embryonic Kidney 293 Cells. The PS133expression plasmid described supra is transfected into HEK293 cells (F.L. Graham et al., J. Gen. Vir. 36:59-72 (1977)). These cells areavailable from the A.T.C.C., 12301 Parklawn Drive, Rockville, Md. 20852,under Accession No. CRL 1573. Transfection is carried out using thecationic lipofectamine-mediated procedure described by P. Hawley-Nelsonet al., Focus 15:73 (1993). Particularly, HEK293 cells are cultured in10 ml DMEM media supplemented with 10% fetal bovine serum (FBS),L-glutamine (2 mM) and freshly seeded into 100 mm culture plates at adensity of 9×10⁶ cells per plate. The cells are grown at 37° C. to aconfluency of between 70% and 80% for transfection. Eight micrograms (8μg) of plasmid DNA is added to 800 μl of Opti-MEM I® medium (Gibco-BRL,Grand Island, N.Y.), and 48-96 μl of Lipofectamine™ Reagent (Gibco-BRL,Grand Island, N.Y.) is added to a second 800 μl portion of Opti-MEM I®media. The two solutions are mixed and incubated at room temperature for15-30 min. After the culture medium is removed from the cells, the cellsare washed once with 10 ml of serum-free DMEM. The Opti-MEMI®-Lipofectamine-plasmid DNA solution is diluted in 6.4 ml of serum-freeDMEM and then overlaid onto the cells. The cells are incubated for 5 hat 37° C., after which time, an additional 8 ml of DMEM with 20% FBS isadded. After 18-24 h, the old medium is aspirated, and the cells areoverlaid with 5 ml of fresh DMEM with 10% FBS. Supernatants and cellextracts are analyzed for PS133 gene activity 72 h after transfection.

[0267] C. Analysis of Prostate Tissue Gene PS133 Antigen Expression. Theculture supernatant, supra, is transferred to cryotubes and stored onice. HEK293 cells are harvested by washing twice with 10 ml coldDulbecco's PBS and lysing by addition of 1.5 ml of CAT lysis buffer(Boehringer Mannheim, Indianapolis, Ind.), followed by incubation for 30min at room temperature. Lysate is transferred to 1.7 ml polypropylenemicrofuge tubes and centrifuged at 1000×g for 10 min. The supernatant istransferred to new cryotubes and stored on ice. Aliquots of cellsupernatants and the lysate of the cells expressing the PS133 proteinconstruct are analyzed for the presence of PS133 recombinant protein.The aliquots can be analyzed using SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), using standard methods and reagents known inthe art. See, e.g., J. Sambrook et al., supra. The gels can then beblotted onto a solid medium such as nitrocellulose, nytran, or the like,and the PS133 protein band can be visualized using western blottingtechniques with anti-myc epitope or anti-histidine monoclonal antibodies(Invitrogen, Carlsbad, Calif.) or PS133 polyclonal serum (see Example14). Alternatively, the expressed PS133 recombinant protein can beanalyzed by mass spectrometry (see Example 12):.

[0268] D. Purification. Purification of the PS133 recombinant proteincontaining the myc-his sequence is performed using the Xpress® affinitychromatography system (Invitrogen, Carlsbad, Calif.) containing anickel-charged agarose resin which specifically binds polyhistidineresidues. Supernatants from 10×100 mm plates, prepared as describedsupra, are pooled and passed over the nickel-charged column. Non-bindingprotein is eluted by washing the column with 50 mM Tris-HCl (pH 7.5)/150mM NaCl buffer, leaving only the myc-his fusion proteins. Bound PS133recombinant protein then is eluted from the column using either anexcess of imidazole or histidine, or a low pH buffer. Alternatively, therecombinant protein can also be purified by binding at the myc-hissequence to an affinity column consisting of either anti-myc oranti-histidine monoclonal antibodies conjugated through a hydrazide orother linkage to an agarose resin and eluting with an excess of mycpeptide or histidine, respectively.

[0269] The purified recombinant protein can then be covalentlycross-linked to a solid phase, such as N-hydroxysuccinimide-activatedsepharose columns (Pharmacia Biotech, Piscataway, N.J.), as directed bysupplier's instructions. These columns containing covalently linkedPS133 recombinant protein, can then be used to purify anti-PS133antibodies from rabbit or mouse sera (see Examples 13 and 14).

[0270] E. Coating Microtiter Plates with PS133 Expressed Proteins.Supernatant from a 100 mm plate, as described supra, is diluted in anappropriate volume of PBS. 100 μl of the resulting mixture is placedinto each well of a Reacti-Bind™ metal chelate microtiter plate (Pierce,Rockford, Ill.), incubated at room temperature while shaking, andfollowed by three washes with 200 μl each of PBS with 0.05% Tween®20.The prepared microtiter plate can then be used to screen polyclonalantisera for the presence of anti-PS133 antibodies (see Example 17).

[0271] Although pcDNA3.1/Myc-His is utilized in this example, it isknown to those skilled in the art that other comparable expressionsystems can be utilized herein with appropriate modifications in reagentand/or techniques and are within the skill of one of ordinary skill inthe art. The largest cloned insert containing the coding region of thePS133 gene is sub-cloned into either (i) a eukaryotic expression vectorwhich may contain, for example, a cytomegalovirus (CMV) promoter and/orprotein fusible sequences which aid in protein expression and detection,or (ii) a bacterial expression vector containing a superoxide-dismutase(SOD) and CMP-KDO synthetase (CKS) or other protein fusion gene forexpression of the protein sequence. Methods and vectors which are usefulfor the production of polypeptides which contain fusion sequences of SODare described in European patent application No. EP 0 196 056, publishedOct. 1, 1986, which is incorporated herein by reference, and vectorscontaining fusion sequences of CKS are described in European patentapplication No. EP 0 331 961, published Sep. 13, 1989, which publicationis also incorporated herein by reference. The purified protein can beused in a variety of techniques, including but not limited to, animalimmunization studies, solid phase immunoassays, etc.

Example 12 Chemical Analysis of Prostate Tissue Proteins

[0272] A. Analysis of Tryptic Peptide Fragments Using MS. Sera frompatients with prostate disease such as prostate cancer, sera frompatients with no prostate disease, extracts of prostate tissues or cellsfrom patients with prostate disease such as prostate cancer, extracts ofprostate tissues or cells from patients with no prostate disease, andextracts of tissues or cells from other non-diseased or diseased organsof patients, are run on a polyacrylamide gel using standard proceduresand stained with Coomassie Blue. Sections of the gel suspected ofcontaining the unknown polypeptide are excised and subjected to anin-gel reduction, acetamidation and tryptic digestion. P. Jeno et al.,Anal. Bio. 224:451-455 (1995) and J. Rosenfeld et al., Anal. Bio.203:173-179 (1992). The gel sections are washed with 100 mM NH₄HCO₃ andacetonitrile. The shrunken gel pieces are swollen in digestion buffer(50 mM NH₄HCO₃, 5 mM CaCl₂ and 12.5 μg/ml trypsin) at 4° C. for 45 min.The supernatant is aspirated and replaced with 5 to 10 μl of digestionbuffer without trypsin and allowed to incubate overnight at 37° C.Peptides are extracted with 3 changes of 5% formic acid and acetonitrileand evaporated to dryness. The peptides are adsorbed to approximately0.1 μl of POROS R2 sorbent (Perseptive Biosystems, Framingham, Mass.)trapped in the tip of a drawn gas chromatography capillary tube bydissolving them in 10 μl of 5% formic acid and passing it through thecapillary. The adsorbed peptides are washed with water and eluted with5% formic acid in 60% methanol. The eluant is passed directly into thespraying capillary of an API III mass spectrometer (Perkin-Elmer Sciex,Thornhill, Ontario, Canada) for analysis by nano-electrospray massspectrometry. M. Wilm et al., Int. J. Mass Spectrom. Ion Process136:167-180 (1994) and M. Wilm et al., Anal. Chem. 66:1-8 (1994). Themasses of the tryptic peptides are determined from the mass spectrumobtained off the first quadrupole. Masses corresponding to predictedpeptides can be further analyzed in MS/MS mode to give the amino acidsequence of the peptide.

[0273] B. Peptide Fragment Analysis Using LC/MS. The presence ofpolypeptides predicted from mRNA sequences found in hyperplastic diseasetissues also can be confirmed using liquid chromatography/tandem massspectrometry (LC/MS/MS). D. Hess et al., METHODS, A Companion to Methodsin Enzymology 6:227-238 (1994). The serum specimen or tumor extract fromthe patient is denatured with SDS and reduced with dithiothreitol (1.5mg/ml) for 30 min at 90° C. followed by alkylation with iodoacetamide (4mg/ml) for 15 min at 25° C. Following acrylamide the polypeptides areelectroblotted to a cationic membrane and stained with Coomassie Blue.Following staining, the membranes are washed and sections thought tocontain the unknown polypeptides are cut out and dissected into smallpieces. The membranes are placed in 500 μl microcentrifuge tubes andimmersed in 10 to 20 μl of proteolytic digestion buffer (100 mMTris-HCl, pH 8.2, containing 0.1 M NaCl, 10% acetonitrile, 2 mM CaCl₂and 5 μg/ml trypsin) (Sigma, St. Louis, Mo.). After 15 h at 37° C., 3 μlof saturated urea and 1 μl of 100 μg/ml trypsin are added and incubatedfor an additional 5 h at 37° C. The digestion mixture is acidified with3 μl of 10% trifluoroacetic acid and centrifuged to separate supernatantfrom membrane. The supernatant is injected directly onto a microbore,reverse phase HPLC column and eluted with a linear gradient ofacetonitrile in 0.05% trifluoroacetic acid. The eluate is fed directlyinto an electrospray mass spectrometer, after passing though a streamsplitter if necessary to adjust the volume of material. The data isanalyzed following the procedures set forth in Example 12, Section A.

Example 13 Gene Immunization Protocol

[0274] A. In Vivo Antigen Expression. Gene immunization circumventsprotein purification steps by directly expressing an antigen in vivoafter inoculation of the appropriate expression vector. Also, productionof antigen by this method may allow correct protein folding andglycosylation since the protein is produced in mammalian tissue. Themethod utilizes insertion of the gene sequence into a plasmid whichcontains a CMV promoter, expansion and purification of the plasmid andinjection of the plasmid DNA into the muscle tissue of an animal.Preferred animals include mice and rabbits. See, for example, H. Daviset al., Human Molecular Genetics 2:1847-1851 (1993). After one or twobooster immunizations, the animal can then be bled, ascites fluidcollected, or the animal's spleen can be harvested for production ofhybridomas.

[0275] B. Plasmid Preparation and Purification. PS133 cDNA sequences aregenerated from the PS133 cDNA-containing vector using appropriate PCRprimers containing suitable 5′ restriction sites following theprocedures described in Example 11. The PCR product is cut withappropriate restriction enzymes and inserted into a vector whichcontains the CMV promoter (for example, pRc/CMV or pcDNA3 vectors fromInvitrogen, San Diego, Calif.). This plasmid then is expanded in theappropriate bacterial strain and purified from the cell lysate using aCsCl gradient or a Qiagen plasmid DNA purification column. All thesetechniques are familiar to one of ordinary skill in the art of molecularbiology.

[0276] C. Immunization Protocol. Anesthetized animals are immunizedintramuscularly with 0.1-100 μg of the purified plasmid diluted in PBSor other DNA uptake enhancers (Cardiotoxin, 25% sucrose). See, forexample, H. Davis et al., Human Gene Therapy 4:733-740 (1993); and P. W.Wolff et al., Biotechniques 11:474-485 (1991). One to two boosterinjections are given at monthly intervals.

[0277] D. Testing and Use of Antiserum. Animals are bled and theresultant sera tested for antibody using peptides synthesized from theknown gene sequence (see Example 16) using techniques known in the art,such as western blotting or EIA techniques. Antisera produced by thismethod can then be used to detect the presence of the antigen in apatient's tissue or cell extract, or in a patient's serum, by ELISA orWestern blotting techniques, such as those described in Examples 15through 18.

Example 14 Production of Antibodies Against PS133

[0278] A. Production of Polyclonal Antisera. Antiserum against PS133 isprepared by injecting appropriate animals with peptides whose sequencesare derived from that of the predicted amino acid sequence of the PS133consensus sequence (SEQUENCE ID NO 7). The synthesis of peptides(SEQUENCE ID NO 25, SEQUENCE ID NO 26, and SEQUENCE ID NO 27) isdescribed in Example 10. Peptides used as immunogen either can beconjugated to a carrier such as keyhole limpet hemocyanine (KLH),prepared as described hereinbelow, or unconjugated (i.e., not conjugatedto a carrier such as KLH).

[0279] 1. Peptide Conjugation. Peptide is conjugated to maleimideactivated keyhole limpet hemocyanine (KLH, commercially available asImject®, available from Pierce Chemical Company, Rockford, Ill.).Imject® contains about 250 moles of reactive maleimide groups per moleof hemocyanine. The activated KLH is dissolved in phosphate bufferedsaline (PBS, pH 8.4) at a concentration of about 7.7 mg/ml. The peptideis conjugated through cysteines occurring in the peptide sequence, or toa cysteine previously added to the synthesized peptide in order toprovide a point of attachment. The peptide is dissolved in dimethylsulfoxide (DMSO, Sigma Chemical Company, St. Louis, Mo.) and reactedwith the activated KLH at a mole ratio of about 1.5 moles of peptide permole of reactive maleimide attached to the KLH. A procedure for theconjugation of peptide (SEQUENCE ID NO 25) is provided hereinbelow. Itis known to the ordinary artisan that the amounts, times and conditionsof such a procedure can be varied to optimize peptide conjugation.

[0280] The conjugation reaction described hereinbelow is based onobtaining 3 mg of KLH peptide conjugate (“conjugated peptide”), whichcontains about 0.77 μmoles of reactive maleimide groups. This quantityof peptide conjugate usually is adequate for one primary injection andfour booster injections for production of polyclonal antisera in arabbit. Briefly, peptide (SEQUENCE ID NO 25) is dissolved in DMSO at aconcentration of 1.16 μmoles/100 μl of DMSO. One hundred microliters(100 μl) of the DMSO solution is added to 380 μl of the activated KLHsolution prepared as described hereinabove, and 20 μl of PBS (pH 8.4) isadded to bring the volume to 500 μl. The reaction is incubated overnightat room temperature with stirring. The extent of reaction is determinedby measuring the amount of unreacted thiol in the reaction mixture. Thedifference between the starting concentration of thiol and the finalconcentration is assumed to be the concentration of peptide which hascoupled to the activated KLH. The amount of remaining thiol is measuredusing Ellman's reagent (5,5′-dithiobis(2-nitrobenzoic acid), PierceChemical Company, Rockford, Ill.). Cysteine standards are made at aconcentration of 0, 0.1, 0.5, 2, 5 and 20 mM by dissolving 35 mg ofcysteine HCl (Pierce Chemical Company, Rockford, Ill.) in 10 ml of PBS(pH 7.2) and diluting the stock solution to the desiredconcentration(s). The photometric determination of the concentration ofthiol is accomplished by placing 200 μl of PBS (pH 8.4) in each well ofan Immulon 2® microwell plate (Dynex Technologies, Chantilly, Va.).Next, 10 μl of standard or reaction mixture is added to each well.Finally, 20 μl of Ellman's reagent at a concentration of 1 mg/ml in PBS(pH 8.4) is added to each well. The wells are incubated for 10 minutesat room temperature, and the absorbance of all wells is read at 415 nmwith a microplate reader (such as the BioRad Model 3550, BioRad,Richmond, Calif.). The absorbance of the standards is used to constructa standard curve and the thiol concentration of the reaction mixture isdetermined from the standard curve. A decrease in the concentration offree thiol is indicative of a successful conjugation reaction. Unreactedpeptide is removed by dialysis against PBS (pH 7.2) at room temperaturefor 6 hours. The conjugate is stored at 2-8° C. if it is to be usedimmediately; otherwise, it is stored at −20° C. or colder.

[0281] 2. Animal Immunization. Female white New Zealand rabbits weighing2 kg or more are used for raising polyclonal antiserum. Generally, oneanimal is immunized per unconjugated or conjugated peptide (prepared asdescribed hereinabove). One week prior to the first immunization, 5 to10 ml of blood is obtained from the animal to serve as a non-immuneprebleed sample.

[0282] Unconjugated or conjugated peptide is used to prepare the primaryimmunogen by emulsifying 0.5 ml of the peptide at a concentration of 2mg/ml in PBS (pH 7.2) which contains 0.5 ml of complete Freund'sadjuvant (CFA) (Difco, Detroit, Mich.). The immunogen is injected intoseveral sites of the animal via subcutaneous, intraperitoneal, and/orintramuscular routes of administration. Four weeks following the primaryimmunization, a booster immunization is administered. The immunogen usedfor the booster immunization dose is prepared by emulsifying 0.5 ml ofthe same unconjugated or conjugated peptide used for the primaryimmunogen, except that the peptide now is diluted to 1 mg/ml with 0.5 mlof incomplete Freund's adjuvant (IFA) (Difco, Detroit, Mich.). Again,the booster dose is administered into several sites and can utilizesubcutaneous, intraperitoneal and intramuscular types of injections. Theanimal is bled (5 ml) two weeks after the booster immunization and theserum is tested for immunoreactivity to the peptide, as described below.The booster and bleed schedule is repeated at 4 week intervals until anadequate titer is obtained. The titer or concentration of antiserum isdetermined by microtiter EIA as described in Example 17, below. Anantibody titer of 1:500 or greater is considered an adequate titer forfurther use and study.

[0283] B. Production of Monoclonal Antibody.

[0284] 1. Immunization Protocol. Mice are immunized using immunogensprepared as described hereinabove, except that the amount of theunconjugated or conjugated peptide for monoclonal antibody production inmice is one-tenth the amount used to produce polyclonal antisera inrabbits. Thus, the primary immunogen consists of 100 μg of unconjugatedor conjugated peptide in 0.1 ml of CFA emulsion; while the immunogenused for booster immunizations consists of 50 μg of unconjugated orconjugated peptide in 0.1 ml of IFA. Hybridomas for the generation ofmonoclonal antibodies are prepared and screened using standardtechniques. The methods used for monoclonal antibody development followprocedures known in the art such as those detailed in Kohler andMilstein, Nature 256:494 (1975) and reviewed in J. G. R. Hurrel, ed.,Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press,Inc., Boca Raton, Fla. (1982). Another method of monoclonal antibodydevelopment which is based on the Kohler and Milstein method is that ofL. T. Mimms et al., Virology 176:604-619 (1990), which is incorporatedherein by reference.

[0285] The immunization regimen (per mouse) consists of a primaryimmunization with additional booster immunizations. The primaryimmunogen used for the primary immunization consists of 100 μg ofunconjugated or conjugated peptide in 50 μl of PBS (pH 7.2) previouslyemulsified in 50 μl of CFA. Booster immunizations performed atapproximately two weeks and four weeks post primary immunization consistof 50 μg of unconjugated or conjugated peptide in 50 μl of PBS (pH 7.2)emulsified with 50 μl IFA. A total of 100 μl of this immunogen isinoculated intraperitoneally and subcutaneously into each mouse.Individual mice are screened for immune response by microtiter plateenzyme immunoassay (EIA) as described in Example 17 approximately fourweeks after the third immunization. Mice are inoculated eitherintravenously, intrasplenically or intraperitoneally with 50 μg ofunconjugated or conjugated peptide in PBS (pH 7.2) approximately fifteenweeks after the third immunization.

[0286] Three days after this intravenous boost, splenocytes are fusedwith, for example, Sp2/0-Ag14 myeloma cells (Milstein Laboratories,England) using the polyethylene glycol (PEG) method. The fusions arecultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 10%fetal calf serum (FCS), plus 1% hypoxanthine, aminopterin and thymidine(HAT). Bulk cultures are screened by mnicrotiter plate EIA following theprotocol in Example 17. Clones reactive with the peptide used animmunogen and non-reactive with other peptides (i.e., peptides of PS133not used as the immunogen) are selected for final expansion. Clones thusselected are expanded, aliquoted and frozen in IMDM containing 10% FCSand 10% dimethyl-sulfoxide.

[0287] 2. Production of Ascites Fluid Containing Monoclonal Antibodies.Frozen hybridoma cells prepared as described hereinabove are thawed andplaced into expansion culture. Viable hybridoma cells are inoculatedintraperitoneally into Pristane treated mice. Ascitic fluid is removedfrom the mice, pooled, filtered through a 0.2μ filter and subjected toan immunoglobulin class G (IgG) analysis to determine the volume of theProtein A column required for the purification.

[0288] 3. Purification of Monoclonal Antibodies From Ascites Fluid.Briefly, filtered and thawed ascites fluid is mixed with an equal volumeof Protein A sepharose binding buffer (1.5 M glycine, 3.0 M NaCl, pH8.9) and refiltered through a 0.2 μ filter. The volume of the Protein Acolumn is determined by the quantity of IgG present in the ascitesfluid. The eluate then is dialyzed against PBS (pH 7.2) overnight at2-8° C. The dialyzed monoclonal antibody is sterile filtered anddispensed in aliquots. The immunoreactivity of the purified monoclonalantibody is confirmed by determining its ability to specifically bind tothe peptide used as the immunogen by use of the EIA microtiter plateassay procedure of Example 17. The specificity of the purifiedmonoclonal antibody is confirmed by determining its lack of binding toirrelevant peptides such as peptides of PS133 not used as the immunogen.The purified anti-PS133 monoclonal thus prepared and characterized isplaced at either 2-8° C. for short term storage or at −80° C. for longterm storage.

[0289] 4. Further Characterization of Monoclonal Antibody. The isotypeand subtype of the monoclonal antibody produced as described hereinabovecan be determined using commercially available kits (available fromAmersham. Inc., Arlington Heights, Ill.). Stability testing also can beperformed on the monoclonal antibody by placing an aliquot of themonoclonal antibody in continuous storage at 2-8° C. and assayingoptical density (OD) readings throughout the course of a given period oftime.

[0290] C. Use of Recombinant Proteins as Immunogens. It is within thescope of the present invention that recombinant proteins made asdescribed herein can be utilized as immunogens in the production ofpolyclonal and monoclonal antibodies, with corresponding changes inreagents and techniques known to those skilled in the art.

Example 15 Purification of Serum Antibodies Which Specifically Bind toPS133 Peptides

[0291] Immune sera, obtained as described hereinabove in Examples 13and/or 14, is affinity purified using immobilized synthetic peptidesprepared as described in Example 10, or recombinant proteins prepared asdescribed in Example 11. An IgG fraction of the antiserum is obtained bypassing the diluted, crude antiserum over a Protein A column (Affi-Gelprotein A, Bio-Rad, Hercules, Calif.). Elution with a buffer (BindingBuffer, supplied by the manufacturer) removes substantially all proteinsthat are not immunoglobulins. Elution with 0.1M buffered glycine (pH 3)gives an immunoglobulin preparation that is substantially free ofalbumin and other serum proteins.

[0292] Immunoaffinity chromatography is performed to obtain apreparation with a higher fraction of specific antigen-binding antibody.The peptide used to raise the antiserum is immobilized on achromatography resin, and the specific antibodies directed against itsepitopes are adsorbed to the resin. After washing away non-bindingcomponents, the specific antibodies are eluted with 0.1 M glycinebuffer, pH 2.3. Antibody fractions are immediately neutralized with 1.0M Tris buffer (pH 8.0) to preserve immunoreactivity. The chromatographyresin chosen depends on the reactive groups present in the peptide. Ifthe peptide has an amino group, a resin such as Affi-Gel 10 or Affi-Gel15 is used (Bio-Rad, Hercules, Calif.). If coupling through a carboxygroup on the peptide is desired, Affi-Gel 102 can be used (Bio-Rad,Hercules, Calif.). If the peptide has a free sulfhydryl group, anorganomercurial resin such as Affi-Gel 501 can be used (Bio-Rad,Hercules, Calif.).

[0293] Alternatively, spleens can be harvested and used in theproduction of hybridomas to produce monoclonal antibodies followingroutine methods known in the art as described hereinabove.

Example 16 Western Blotting of Tissue Samples

[0294] Protein extracts are prepared by homogenizing tissue samples in0.1 M Tris-HCl (pH 7.5), 15% (w/v) glycerol, 0.2 mM EDTA, 1.0 mM1,4-dithiothreitol, 10 μg/ml leupeptin and 1.0 mMphenylmethylsulfonylfluoride (Kain et al., Biotechniques, 17:982(1994)). Following homogenization, the homogenates are centrifuged at 4°C. for 5 minutes to separate supernate from debris. For proteinquantitation, 3-10 μl of supernate are added to 1.5 ml of bicinchoninicacid reagent (Sigma, St. Louis, Mo.), and the resulting absorbance at562 nm is measured.

[0295] For SDS-PAGE, samples are adjusted to desired proteinconcentration with Tricine Buffer (Novex, San Diego, Calif.), mixed withan equal volume of 2X Tricine sample buffer (Novex, San Diego, Calif.),and heated for 5 minutes at 100° C. in a thermal cycler. Samples arethen applied to a Novex 10-20% Precast Tricine Gel for electrophoresis.Following electrophoresis, samples are transferred from the gels tonitrocellulose membranes in Novex Tris-Glycine Transfer buffer.Membranes are then probed with specific anti-peptide antibodies usingthe reagents and procedures provided in the Western Lights or WesternLights Plus (Tropix, Bedford, Mass.) chemiluminesence detection kits.Chemiluminescent bands are visualized by exposing the developedmembranes to Hyperfilm ECL (Amersham, Arlington Heights, Ill.).

[0296] Competition experiments are carried out in an analogous manner asabove, with the following exception; the primary antibodies(anti-peptide polyclonal antisera) are pre-incubated for 30 minutes atroom temperature with varying concentrations of peptide immunogen priorto exposure to the nitrocellulose filter. Development of the Western isperformed as above.

[0297] After visualization of the bands on film, the bands can also bevisualized directly on the membranes by the addition and development ofa chromogenic substrate such as 5-bromo-4-chloro-3-indolyl phosphate(BCIP). This chromogenic solution contains 0.016% BCIP in a solutioncontaining 100 mM NaCl, 5 mM MgCl₂ and 100 mM Tris-HCl (pH 9.5). Thefilter is incubated in the solution at room temperature until the bandsdevelop to the desired intensity. Molecular mass determination is madebased upon the mobility of pre-stained molecular weight standards(Novex, San Diego, Calif.) or biotinylated molecular weight standards(Tropix, Bedford, Mass.).

Example 17 EIA Microtiter Plate Assay

[0298] The immunoreactivity of antiserum preferably obtained fromrabbits or mice as described in Example 13 or Example 14 is determinedby means of a microtiter plate EIA, as follows. Synthetic peptidesprepared as described in Example 10 or recombinant proteins prepared asdescribed in Example 11 are dissolved in 50 mM carbonate buffer (pH 9.6)to a final concentration of 2 μg/ml. Next, 100 μl of the peptide orprotein solution is placed in each well of an Immulon 2® microtiterplate (Dynex Technologies, Chantilly, Va.). The plate is incubatedovernight at room temperature and then washed four times with deionizedwater. The wells are blocked by adding 125 μl of a suitable proteinblocking agent, such as Superblock® (Pierce Chemical Company, Rockford,Ill.), in phosphate buffered saline (PBS, pH 7.4) to each well and thenimmediately discarding the solution. This blocking procedure isperformed three times. Antiserum obtained from immunized rabbits or miceprepared as previously described is diluted in a protein blocking agent(e.g., a 3% Superblock® solution) in PBS containing 0.05% Tween-20®(monolaurate polyoxyethylene ether) (Sigma Chemical Company, St. Louis,Mo.) and 0.05% sodium azide at dilutions of 1:500, 1:2500, 1:12,500,1:62,500 and 1:312,500 and placed in each well of the coated microtiterplate. The wells then are incubated for three hours at room temperature.Each well is washed four times with deionized water. One hundred μl ofalkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouseIgG antiserum (Southern Biotech, Birmingham, Ala.), diluted 1:2000 in 3%Superblock® solution in phosphate buffered saline containing 0.05% Tween20® and 0.05% sodium azide, is added to each well . The wells areincubated for two hours at room temperature. Next, each well is washedfour times with deionized water. One hundred microliters (100 μl) ofparanitrophenyl phosphate substrate (Kirkegaard and Perry Laboratories,Gaithersburg, Md.) then is added to each well. The wells are incubatedfor thirty minutes at room temperature. The absorbance at 405 nm is readof each well. Positive reactions are identified by an increase inabsorbance at 405 nm in the test well above that absorbance given by anon-immune serum (negative control). A positive reaction is indicativeof the presence of detectable anti-PS133 antibodies.

[0299] In addition to titers, apparent affinities [K_(d)(app)] may alsobe determined for some of the anti-peptide antisera. EIA microtiterplate assay results can be used to derive the apparent dissociationconstants (K_(d)) based on an analog of the Michaelis-Menten equation(V. Van Heyningen, Methods in Enzymology, Vol. 121, p. 472 (1986) andfurther described in X. Qiu et al., Journal of Immunology, Vol. 156, p.3350 (1996)):$\left\lbrack {{Ag} - {Ab}} \right\rbrack = {\left\lbrack {{Ag} - {Ab}} \right\rbrack_{\max}X\frac{\lbrack{Ab}\rbrack}{\lbrack{Ab}\rbrack = K_{d}}}$

[0300] Where [Ag-Ab] is the antigen-antibody complex concentration,[Ag-Ab]_(max) is the maximum complex concentration, [Ab] is the antibodyconcentration, and K_(d) is the dissociation constant. During the curvefitting, the [Ag-Ab] is replaced with the background subtracted value ofthe OD_(405nm) at the given concentration of Ab. Both K_(d) and[OD_(405nm)]_(max), which corresponds to the [Ag-Ab]_(max), are treatedas fitted parameters. The software program Origin can be used for thecurve fitting.

Example 18 Coating of Solid Phase Particles

[0301] A. Coating of Microparticles with Antibodies Which SpecificallyBind to PS133 Antigen. Affinity purified antibodies which specificallybind to PS133 protein (see Example 15) are coated onto microparticles ofpolystyrene, carboxylated polystyrene, polymethylacrylate or similarparticles having a radius in the range of about 0.1 to 20 μm.Microparticles may be either passively or actively coated. One coatingmethod comprises coating EDAC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (AldrichChemical Co., Milwaukee, Wis.) activated carboxylated latexmicroparticles with antibodies which specifically bind to PS133 protein,as follows. Briefly, a final 0.375% solid suspension of resin washedcarboxylated latex microparticles (available from Bangs Laboratories,Carmel, Ind. or Serodyn, Indianapolis, Ind.) are mixed in a solutioncontaining 50 mM MES buffer, pH 4.0 and 150 mg/l of affinity purifiedanti-PS133 antibody (see Example 14) for 15 min in an appropriatecontainer. EDAC coupling agent is added to a final concentration of 5.5μg/ml to the mixture and mixed for 2.5 h at room temperature.

[0302] The microparticles then are washed with 8 volumes of a Tween20®/sodium phosphate wash buffer (pH 7.2) by tangential flow filtrationusing a 0.2 μm Microgon Filtration module. Washed microparticles arestored in an appropriate buffer which usually contains a dilutesurfactant and irrelevant protein as a blocking agent, until needed.

[0303] B. Coating of ¼ Inch Beads. Antibodies which specifically bind toPS133-antigen also may be coated on the surface of ¼ inch polystyrenebeads by routine methods known in the art (Snitman et al., U.S. Pat. No.5,273,882, incorporated herein by reference) and used in competitivebinding or EIA sandwich assays.

[0304] Polystyrene beads first are cleaned by ultrasonicating them forabout 15 seconds in 10 mM NaHCO₃ buffer at pH 8.0. The beads then arewashed in deionized water until all fines are removed. Beads then areimmersed in an antibody solution in 10 mM carbonate buffer, pH 8 to 9.5.The antibody solution can be as dilute as 1 μg/ml in the case of highaffinity monoclonal antibodies or as concentrated as about 500 μg/ml forpolyclonal antibodies which have not been affinity purified. Beads arecoated for at least 12 hours at room temperature, and then they arewashed with deionized water. Beads may be air dried or stored wet (inPBS, pH 7.4). They also may be overcoated with protein stabilizers (suchas sucrose) or protein blocking agents used as non-specific bindingblockers (such as irrelevant proteins, Carnation skim milk, Superblock®,or the like).

Example 19 Microparticle Enzyme Immunoassay (MEIA)

[0305] PS133 antigens are detected in patient test samples by performinga standard antigen competition EIA or antibody sandwich EIA andutilizing a solid phase such as microparticles (MEIA). The assay can beperformed on an automated analyzer such as the IMx® Analyzer (AbbottLaboratories, Abbott Park, Ill.).

[0306] A. Antibody Sandwich EIA. Briefly, samples suspected ofcontaining PS133 antigen are incubated in the presence of anti-PS133antibody-coated microparticles (prepared as described in Example 17) inorder to form antigen/antibody complexes. The microparticles then arewashed and an indicator reagent comprising an antibody conjugated to asignal generating compound (i.e., enzymes such as alkaline phosphataseor horseradish peroxide) is added to the antigen/antibody complexes orthe microparticles and incubated. The microparticles are washed and thebound antibody/antigen/antibody complexes are detected by adding asubstrate (e.g., 4-methyl umbelliferyl phosphate (MUP), or OPD/peroxide,respectively), that reacts with the signal generating compound togenerate a measurable signal. An elevated signal in the test sample,compared to the signal generated by a negative control, detects thepresence of PS133 antigen. The presence of PS133 antigen in the testsample is indicative of a diagnosis of a prostate disease or condition,such as prostate cancer.

[0307] B. Competitive Binding Assay. The competitive binding assay usesa peptide or protein-that generates a measurable signal when the labeledpeptide is contacted with an anti-peptide antibody coated microparticle.This assay can be performed on the IMx® Analyzer (available from AbbottLaboratories, Abbott Park, Ill.). The labeled peptide is added to thePS133 antibody-coated microparticles (prepared as described in Example17) in the presence of a test sample suspected of containing PS133antigen, and incubated for a time and under conditions sufficient toform labeled PS133 peptide (or labeled protein)/bound antibody complexesand/or patient PS133 antigen/bound antibody complexes. The PS133 antigenin the test sample competes with the labeled PS133 peptide (or PS133protein) for binding sites on the microparticle. PS133 antigen in thetest sample results in a lowered binding of labeled peptide and antibodycoated microparticles in the assay since antigen in the test sample andthe PS133 peptide or PS133 protein compete for antibody binding sites. Alowered signal (compared to a control) indicates the presence of PS133antigen in the test sample. The presence of PS133 antigen suggests thediagnosis of a prostate disease or condition, such as prostate cancer.

Example 20 Identifying Inhibitors of PS133 Proteolytic Activity

[0308] Inhibitors of the biological (catalytic) activity of PS133polypeptides are discovered using peptide-based substrates which areconjugated with a chromogenic or fluorogenic tag. Such a peptidesubstrate has the following structure:

[0309] X—(Y)_(n)-Z, wherein:

[0310] X represents an appropriate amino protecting group, such asacetyl or benzyloxycarbonyl;

[0311] Y is any naturally or non-naturally occurring amino acid thatforms a substrate capable of being recognized and cleaved by a PS133polypeptide in the absence of an inhibitor;

[0312] n represents an integer which is any number, usually less than20; and

[0313] Z represents any detectable tag such as a chromogenic orfluorogenic tag (e.g., para-nitroanilide or N-methyl coumarin), which,upon cleavage of the Y group from the above-described peptide substrate,undergoes a change in a signal characteristic (e.g., intensity, color,wavelength, or the like) which can thus be monitored as an indicator ofthe presence or absence of inhibitory activity.

[0314] An assay is established in which PS133 polypeptide-catalyzedcleavage of the peptide substrate is monitored in the presence orabsence of a potential inhibitory compound or compound mixture. If thepotential inhibitor does not inhibit the PS133 polypeptide proteolyticactivity and the substrate (Y-group) is cleaved, the detectable tag Zundergoes a corresponding change in configuration. This configurationchange allows a change in fluorescence to be detected by a fluorimeterin the case of a fluorogenic tag, or a change in color to be detected bya spectrophotometer in the case of a chromogenic tag. When the inhibitorsuccessfully inhibits PS133 protease from cleaving the substrate, the Ygroup is not cleaved and Z does not have a change in configuration.Therefore, no fluorescence or color change is detectable, indicatingthat the compound has inhibited the action of the PS133 polypeptide. Theassay described herein is also used to discover agonists/antagonists ofproteolytic action of PS133. Because the PS133 polypeptide possesses anAsp residue at the base of the specificity pocket at position 165 (seeFIG. 3), preferred amino acids at peptide substrate position Y1 are Argand Lys, in analogy with structural features of Asp 189 of the relatedenzyme human trypsin and its corresponding peptide substrate Y1 residuepreference (as discussed in C. Gaboriaud et al., J. Mol. Bio.,259:995-1010 (1997), Academic Press, Ltd.).

[0315] The PS133 polynucleotides and the proteins encoded thereby whichare provided and discussed hereinabove are useful as markers of prostatetissue disease, especially prostate cancer. Tests based upon theappearance of this marker in a test sample such as blood, plasma orserum can provide low cost, non-invasive, diagnostic information to aidthe physician to make a diagnosis of cancer, to help select a therapyprotocol, or to monitor the success of a chosen therapy. This marker mayappear in readily accessible body fluids such as blood, urine or stoolas antigens derived from the diseased tissue which are detectable byimmunological methods. This marker may be elevated in a disease state,altered in a disease state, or be a normal protein of the prostate whichappears in an inappropriate body compartment.

1 76 81 base pairs nucleic acid single linear 1 CTGCTGTAGC TGCCGCCACTGCCGTNTCCG NCGNCANTGG GNCCCCAGAG CCCCAGCCCC 60 AGAGCCTAGG AACCTGGGGC C81 222 base pairs nucleic acid single linear 2 GCTGCCGCCA CTGCCGTCTCCGCCGCCACT GGGCCCCCNG AGCCCCAGCN CCAGAGCCTA 60 GGAACCTGGG GCCCGCTCCTCCCCCCTCCA GGCCATGAGG ATTCTGCAGT TAATCCTGCT 120 TGCTCTGGCA ACAGGGCTTGTAGGGGGAGA GACCAGGATC ATCAAGGGGT TCGAGTGCNA 180 GCCTCACTCC CAGCCCTGGCAGGCAGCCCT GTTCGAGAAA AC 222 239 base pairs nucleic acid single linear 3CCCAGCCCTG GCAGGCAGCC CTGTTCAAGA AGACGCGGCT ACTCTGTGGG GCGACGCTCA 60TCGCCCCCAG ATGGCTCCTG ACAGCAGCCC ACTGCCTCAA GCCCCGCTAC ATAGTTCACC 120TGGGGCAGCA CAACCTCCAG AAGGAGGAGG GCTGTGAGCA GACCCGGACA GCCACTGAGT 180CCTTCCCCCA CCCCGGCTTC AACAACAGCC TCCCCAACAA AGACCACCGC AATGACATC 239 250base pairs nucleic acid single linear 4 GACCACCGCA ATGACATCAT GCTGGTGAAGATGGCATCGC CAGTCTCCAT CACCTGGGCT 60 GTGCGACCCC TCACCCTCTC CTCACGCTGTGTCACTGCTG GCACCAGCTG CCTCATTTCC 120 GGCTGGGGCA GCACGTCCAG CCCCCAGTTACGCCTGCCTC ACACCTTGCG ATGCGCCAAC 180 ATCACCATCA TTGAGCACCA GAAGTGTGAGAACGCCTACC CCGGCAACAT CACAGACACC 240 ATGGTGTGTG 250 262 base pairsnucleic acid single linear 5 AACGCCTACC CCGGCAACAT CACAGACACC ATGGTGTGTGCCAGCGTGCA GGAAGGGGGC 60 AAGGACTCCT GCCAGGGTGA CTCCGGGGGC CCTCTGGTCTGTAACCAGTC TCTTCAAGGC 120 ATTATCTCCT GGGGCCAGGA TCCGTGTGCG ATCACCCGAAAGCCTGGTGT CTACACGAAA 180 GTCTGCAAAT ATGTGGACTG GATCCAGGAG ACGATGAAGAACAATTAGAC TGGACCCACC 240 CACCACAGCC CATCACCCTC CA 262 406 base pairsnucleic acid single linear 6 CATTTAGCAA ATATTTATTG AAACCTTGAT ATATGGCCAGGAGCTGTCTT TCGGGGCTGG 60 GGATACAACA GAGAACAAAC CAGGTGTTGT CATTCCCAGAGTCACAATAT TTCAAGGCAG 120 AATTTGAATC CAGGTCTCAC TNGATTTCGA ACCCCAGGTTCGATTATTAA GTGACAGCAT 180 CTCCTGTAGT CCAGGAGGCC CAAAGAATGT TCGCAGAGGGTCTTGGCTTA GGGTTTCTTA 240 TTAACAGAGT GAACAGGAAC CAAACACCAA GTGGAAATGGAGGGTGATGG GCTGTGGTGG 300 GTGGGTCCAG TCTAATTGTT CTTCATCGTC TCCTGGATCCAGTCCACATA TTTGCAGACT 360 TTCGTGTAGA CACCAGGCTT TCGGGTGATC GCACACGGATCCTGGC 406 1166 base pairs nucleic acid single linear 7 CTGCTGTAGCTGCCGCCACT GCCGTCTCCG CCGCCACTGG GCCCCCAGAG CCCCAGCCCC 60 AGAGCCTAGGAACCTGGGGC CCGCTCCTCC CCCCTCCAGG CCATGAGGAT TCTGCAGTTA 120 ATCCTGCTTGCTCTGGCAAC AGGGCTTGTA GGGGGAGAGA CCAGGATCAT CAAGGGGTTC 180 GAGTGCNAGCCTCACTCCCA GCCCTGGCAG GCAGCCCTGT TCRAGAARAC GCGGCTACTC 240 TGTGGGGCGACGCTCATCGC CCCCAGATGG CTCCTGACAG CAGCCCACTG CCTCAAGCCC 300 CGCTACATAGTTCACCTGGG GCAGCACAAC CTCCAGAAGG AGGAGGGCTG TGAGCCAGAC 360 CGGACAGCCACTGAGTCCTT CCCCCACCCC GGCTTCAACA ACAGCCTCCC CAACAAAGAC 420 CACCGCAATGACATCATGCT GGTGAAGATG GCATCGCCAG TCTCCATCAC CTGGGCTGTG 480 CGACCCCTCACCCTCTCCTC ACGCTGTGTC ACTGCTGGCA CCAGCTGCCT CATTTCCATG 540 TGGGGCAGCACGTCCAGCCC CCAGTTACGC CTGCCTCACA CCTTGCGATG CGCCAACATC 600 ACCATCATTGAGCACCAGAA GTGTGAGAAC GCCTACCCCG GCAACATCAC AGACACCATG 660 GTGTGTGCCAGCGTGCAGGA AGGGGGCAAG GACTCCTGCC AGGGTGACTC CGGGGGCCCT 720 CTGGTCTGTAACCAGTCTCT TCAAGGCATT ATCTCCTGGG GCCAGGATCC GTGTGCGATC 780 ACCCGAAAGCCTGGTGTCTA CACGAAAGTC TGCAAATATG TGGACTGGAT CCAGGAGACG 840 ATGAAGAACAATTAGACTGG ACCCACCCAC CACAGCCCAT CACCCTCCAT TTCCACTTGG 900 TGTTTGGTTCCTGTTCACTC TGTTAATAAG AAACCCTAAG CCAAGACCCT CTGCGAACAT 960 TCTTTGGGCCTCCTGGACTA CAGGAGATGC TGTCACTTAA TAATCGAACC TGGGGTTCGA 1020 AATCNAGTGAGACCTGGATT CAAATTCTGC CTTGAAATAT TGTGACTCTG GGAATGACAA 1080 CACCTGGTTTGTTCTCTGTT GTATCCCCAG CCCCGAAAGA CAGCTCCTGG CCATATATCA 1140 AGGTTTCAATAAATATTTGC TAAATG 1166 1192 base pairs nucleic acid single linear cDNA 8GAATTCGAAT TCGCTGCCGC CACTGCCGTC TCCGCCGCCA CTGGGCCCCC AGAGCCCCAG 60CCCCAGAGCC TAGGAACCTG GGGCCCGCTC CTCCCCCCTC CAGGCCATGA GGATTCTGCA 120GTTAATCCTG CTTGCTCTGG CAACAGGGCT TGTAGGGGGA GAGACCAGGA TCATCAAGGG 180GTTCGAGTGC AAGCCTCACT CCCAGCCCTG GCAGGCAGCC CTGTTCGAGA AGACGCGGCT 240ACTCTGTGGG GCGACGCTCA TCGCCCCCAG ATGGCTCCTG ACAGCAGCCC ACTGCCTCAA 300GCCCCGCTAC ATAGTTCACC TGGGGCAGCA CAACCTCCAG AAGGAGGAGG GCTGTGAGCA 360GACCCGGACA GCCACTGAGT CCTTCCCCCA CCCCGGCTTC AACAACAGCC TCCCCAACAA 420AGACCACCGC AATGACATCA TGCTGGTGAA GATGGCATCG CCAGTCTCCA TCACCTGGGC 480TGTGCGACCC CTCACCCTCT CCTCACGCTG TGTCACTGCT GGCACCAGCT GCCTCATTTC 540CGGCTGGGGC AGCACGTCCA GCCCCCAGTT ACGCCTGCCT CACACCTTGC GATGCGCCAA 600CATCACCATC ATTGAGCACC AGAAGTGTGA GAACGCCTAC CCCGGCAACA TCACAGACAC 660CATGGTGTGT GCCAGCGTGC AGGAAGGGGG CAAGGACTCC TGCCAGGGTG ACTCCGGGGG 720CCCTCTGGTC TGTAACCAGT CTCTTCAAGG CATTATCTCC TGGGGCCAGG ATCCGTGTGC 780GATCACCCGA AAGCCTGGTG TCTACACGAA AGTCTGCAAA TATGTGGACT GGATCCAGGA 840GACGATGAAG AACAATTAGA CTGGACCCAC CCACCACAGC CCATCACCCT CCATTTCCAC 900TTGGTGTTTG GTTCCTGTTC ACTCTGTTAA TAAGAAACCC TAAGCCAAGA CCCTCTACGA 960ACATTCTTTG GGCCTCCTGG ACTACAGGAG ATGCTGTCAC TTAATAATCA ACCTGGGGTT 1020CGAAATCAGT GAGACCTGGA TTCAAATTCT GCCTTGAAAT ATTGTGACTC TGGGAATGAC 1080AACACCTGGT TTGTTCTCTG TTGTATCCCC AGCCCCAAAG ACAGCTCCTG GCCATATATC 1140AAGGTTTCAA TAAATATTTG CTAAATGAAA AAAAAAAAAA AAGGGCGGCC GC 1192 68 basepairs nucleic acid single linear 9 AGCTCGGAAT TCCGAGCTTG GATCCTCTAGAGCGGCCGCC GACTAGTGAG CTCGTCGACC 60 CGGGAATT 68 68 base pairs nucleicacid single linear 10 AATTAATTCC CGGGTCGACG AGCTCACTAG TCGGCGGCCGCTCTAGAGGA TCCAAGCTCG 60 GAATTCCG 68 24 base pairs nucleic acid singlelinear 11 AGCGGATAAC AATTTCACAC AGGA 24 18 base pairs nucleic acidsingle linear 12 TGTAAAACGA CGGCCAGT 18 18 base pairs nucleic acidsingle linear 13 TCAAGCCCCG CTACATAG 18 19 base pairs nucleic acidsingle linear 14 CACCAGAAGT GTGAGAACG 19 18 base pairs nucleic acidsingle linear 15 ACCCTCCATT TCCACTTG 18 18 base pairs nucleic acidsingle linear 16 ATCAAGGGGT TCGAGTGC 18 21 base pairs nucleic acidsingle linear 17 CCTGGTGTCT ACACGAAAGT C 21 18 base pairs nucleic acidsingle linear 18 CCCAGGAGAT AATGCCTT 18 18 base pairs nucleic acidsingle linear 19 CCAGCATGAT GTCATTGC 18 18 base pairs nucleic acidsingle linear 20 CTGTTGCCAG AGCAAGCA 18 22 base pairs nucleic acidsingle linear 21 AGTGACAGCA TCTCCTGTAG TC 22 20 base pairs nucleic acidsingle linear 22 GGTAGGCGTT CTCACACTTC 20 21 base pairs nucleic acidsingle linear 23 CAGGTGTTGT CATTCCCAGA G 21 248 amino acids amino acidsingle linear None 24 Met Arg Ile Leu Gln Leu Ile Leu Leu Ala Leu AlaThr Gly Leu Val 1 5 10 15 Gly Gly Glu Thr Arg Ile Ile Lys Gly Phe GluCys Pro His Ser Gln 20 25 30 Pro Trp Gln Ala Ala Leu Phe Lys Thr Arg LeuLeu Cys Gly Ala Thr 35 40 45 Leu Ile Ala Pro Arg Trp Leu Leu Thr Ala AlaHis Cys Leu Lys Pro 50 55 60 Arg Tyr Ile Val His Leu Gly Gln His Asn LeuGln Lys Glu Glu Gly 65 70 75 80 Cys Glu Gln Thr Arg Thr Ala Thr Glu SerPhe Pro His Pro Gly Phe 85 90 95 Asn Asn Ser Leu Pro Asn Lys Asp His ArgAsn Asp Ile Met Leu Val 100 105 110 Lys Met Ala Ser Pro Val Ser Ile ThrTrp Ala Val Arg Pro Leu Thr 115 120 125 Leu Ser Ser Arg Cys Val Thr AlaGly Thr Ser Cys Leu Ile Ser Gly 130 135 140 Trp Gly Ser Thr Ser Ser ProGln Leu Arg Leu Pro His Thr Leu Arg 145 150 155 160 Cys Ala Asn Ile ThrIle Ile Glu His Gln Lys Cys Glu Asn Ala Tyr 165 170 175 Pro Gly Asn IleThr Asp Thr Met Val Cys Ala Ser Val Gln Glu Gly 180 185 190 Gly Lys AspSer Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Asn 195 200 205 Gln SerLeu Gln Gly Ile Ile Ser Trp Gly Gln Asp Pro Cys Ala Ile 210 215 220 ThrArg Lys Pro Gly Val Tyr Thr Lys Val Cys Lys Tyr Val Asp Trp 225 230 235240 Ile Gln Glu Thr Met Lys Asn Asn 245 35 amino acids amino acid singlelinear None 25 Asn Leu Gln Lys Glu Glu Gly Cys Glu Gln Thr Arg Thr AlaThr Glu 1 5 10 15 Ser Phe Pro His Pro Gly Phe Asn Asn Ser Leu Pro AsnLys Asp His 20 25 30 Arg Asn Asp 35 36 amino acids amino acid singlelinear None 26 Glu His Gln Lys Cys Glu Asn Ala Tyr Pro Gly Asn Ile ThrAsp Thr 1 5 10 15 Met Val Cys Ala Ser Val Gln Glu Gly Gly Lys Asp SerCys Gln Gly 20 25 30 Asp Ser Gly Gly 35 33 amino acids amino acid singlelinear None 27 Ser Trp Gly Gln Asp Pro Cys Ala Ile Thr Arg Lys Pro GlyVal Tyr 1 5 10 15 Thr Lys Val Cys Lys Tyr Val Asp Trp Ile Gln Glu ThrMet Lys Asn 20 25 30 Asn 251 amino acids amino acid single linear None28 Ile Ile Gly Gly Ser Asp Ala Asp Ile Lys Asn Phe Pro Trp Gln Val 1 510 15 Phe Phe Asp Asn Pro Trp Ala Gly Gly Ala Leu Ile Asn Glu Tyr Trp 2025 30 Val Leu Thr Ala Ala His Val Val Glu Gly Asn Arg Glu Pro Thr Met 3540 45 Tyr Val Gly Ser Thr Ser Val Gln Thr Ser Arg Leu Ala Lys Ser Lys 5055 60 Met Leu Thr Pro Glu His Val Phe Ile His Pro Gly Trp Lys Leu Leu 6570 75 80 Glu Val Pro Glu Gly Arg Thr Asn Phe Asp Asn Asp Ile Ala Leu Val85 90 95 Arg Leu Lys Asp Pro Val Lys Met Gly Pro Thr Val Ser Pro Ile Cys100 105 110 Leu Pro Gly Thr Ser Ser Asp Tyr Asn Leu Met Asp Gly Asp LeuGly 115 120 125 Leu Ile Ser Gly Trp Gly Arg Thr Glu Lys Arg Asp Arg AlaVal Arg 130 135 140 Leu Lys Ala Ala Arg Leu Pro Val Ala Pro Leu Arg LysCys Lys Glu 145 150 155 160 Val Lys Val Glu Lys Pro Thr Ala Asp Ala GluAla Tyr Val Phe Thr 165 170 175 Pro Asn Met Ile Cys Ala Gly Gly Glu LysGly Met Asp Ser Cys Lys 180 185 190 Gly Asp Ser Gly Gly Ala Phe Ala ValGln Asp Pro Asn Asp Lys Thr 195 200 205 Lys Phe Tyr Ala Ala Gly Leu ValSer Trp Gly Pro Gln Cys Gly Thr 210 215 220 Tyr Gly Leu Tyr Thr Arg ValLys Asn Tyr Val Asp Trp Ile Met Lys 225 230 235 240 Thr Met Gln Glu AsnSer Thr Pro Arg Glu Asp 245 250 242 amino acids amino acid single linearNone 29 Ile Ile Gly Gly Gln Lys Ala Lys Met Gly Asn Phe Pro Trp Gln Val1 5 10 15 Phe Thr Asn Ile His Gly Arg Gly Gly Gly Ala Leu Leu Gly AspArg 20 25 30 Trp Ile Leu Thr Ala Ala His Thr Leu Tyr Pro Lys Glu His GluAla 35 40 45 Gln Ser Asn Ala Ser Leu Asp Val Phe Leu Gly His Thr Asn ValGlu 50 55 60 Glu Leu Met Lys Leu Gly Asn His Pro Ile Arg Arg Val Ser ValHis 65 70 75 80 Pro Asp Tyr Arg Gln Asp Glu Ser Tyr Asn Phe Glu Gly AspIle Ala 85 90 95 Leu Leu Glu Leu Glu Asn Ser Val Thr Leu Gly Pro Asn LeuLeu Pro 100 105 110 Ile Cys Leu Pro Asp Asn Asp Thr Phe Tyr Asp Leu GlyLeu Met Gly 115 120 125 Tyr Val Ser Gly Phe Gly Val Met Glu Glu Lys IleAla His Asp Leu 130 135 140 Arg Phe Val Arg Leu Pro Val Ala Asn Pro GlnAla Cys Glu Asn Trp 145 150 155 160 Leu Arg Gly Lys Asn Arg Met Asp ValPhe Ser Gln Asn Met Phe Cys 165 170 175 Ala Gly His Pro Ser Leu Lys GlnAsp Ala Cys Gln Gly Asp Ser Gly 180 185 190 Gly Val Phe Ala Val Arg AspPro Asn Thr Asp Arg Trp Val Ala Thr 195 200 205 Gly Ile Val Ser Trp GlyIle Gly Cys Ser Arg Gly Tyr Gly Phe Tyr 210 215 220 Thr Lys Val Leu AsnTyr Val Asp Trp Ile Lys Lys Glu Met Glu Glu 225 230 235 240 Glu Asp 229amino acids amino acid single linear None 30 Ile Val Gly Gly His Glu AlaGln Pro His Ser Arg Pro Tyr Met Ala 1 5 10 15 Ser Leu Gln Met Arg GlyAsn Pro Gly Ser His Phe Cys Gly Gly Thr 20 25 30 Leu Ile His Pro Ser PheVal Leu Thr Ala Pro His Cys Leu Arg Asp 35 40 45 Ile Pro Gln Arg Leu ValAsn Val Val Leu Gly Ala His Asn Val Arg 50 55 60 Thr Gln Glu Pro Thr GlnGln His Phe Ser Val Ala Gln Val Phe Leu 65 70 75 80 Asn Asn Tyr Asp AlaGlu Asn Lys Leu Asn Asp Ile Leu Leu Ile Gln 85 90 95 Leu Ser Ser Pro AlaAsn Leu Ser Ala Ser Val Thr Ser Val Gln Leu 100 105 110 Pro Gln Gln AspGln Pro Val Pro His Gly Thr Gln Cys Leu Ala Met 115 120 125 Gly Trp GlyArg Val Gly Ala His Asp Pro Pro Ala Gln Val Leu Gln 130 135 140 Glu LeuAsn Val Thr Val Val Thr Phe Phe Cys Arg Pro His Asn Ile 145 150 155 160Cys Thr Phe Val Pro Arg Arg Lys Ala Gly Ile Cys Phe Gly Asp Ser 165 170175 Gly Gly Pro Leu Ile Cys Asp Gly Ile Ile Gln Gly Ile Asp Ser Phe 180185 190 Val Ile Trp Gly Cys Ala Thr Arg Leu Phe Pro Asp Phe Phe Thr Arg195 200 205 Val Ala Leu Tyr Val Asp Trp Ile Arg Ser Thr Leu Arg Arg ValGlu 210 215 220 Ala Lys Gly Arg Pro 225 238 amino acids amino acidsingle linear None 31 Ile Val Gly Gly Arg Arg Ala Arg Pro His Ala TrpPro Phe Met Val 1 5 10 15 Ser Leu Gln Leu Arg Gly Gly His Phe Cys GlyAla Thr Leu Ile Ala 20 25 30 Pro Asn Phe Val Met Ser Ala Ala His Cys ValAla Asn Val Asn Val 35 40 45 Arg Ala Val Arg Val Val Leu Gly Ala His AsnLeu Ser Arg Arg Glu 50 55 60 Pro Thr Arg Gln Val Phe Ala Val Gln Arg IlePhe Glu Asn Gly Tyr 65 70 75 80 Asp Pro Val Asn Leu Leu Asn Asp Ile ValIle Leu Gln Leu Asn Gly 85 90 95 Ser Ala Thr Ile Asn Ala Asn Val Gln ValAla Gln Leu Pro Ala Gln 100 105 110 Gly Arg Arg Leu Gly Asn Gly Val GlnCys Leu Ala Met Gly Trp Gly 115 120 125 Leu Leu Gly Arg Asn Arg Gly IleAla Ser Val Leu Gln Glu Leu Asn 130 135 140 Val Thr Val Val Thr Ser LeuCys Arg Arg Ser Asn Val Cys Thr Leu 145 150 155 160 Val Arg Gly Arg GlnAla Gly Val Cys Phe Gly Asp Ser Gly Ser Pro 165 170 175 Leu Val Cys AsnGly Leu Ile His Gly Ile Ala Ser Phe Val Arg Gly 180 185 190 Gly Cys AlaSer Gly Leu Tyr Pro Asp Ala Phe Ala Pro Val Ala Gln 195 200 205 Phe ValAsn Trp Ile Asp Ser Ile Ile Gln Arg Ser Glu Asp Asn Pro 210 215 220 CysPro His Pro Arg Asp Pro Asp Pro Ala Ser Arg Thr His 225 230 235 225amino acids amino acid single linear None 32 Ile Val Gly Gly Arg Lys AlaArg Pro Arg Gln Phe Pro Phe Leu Ala 1 5 10 15 Ser Ile Gln Asn Gln GlyArg His Phe Cys Gly Gly Ala Leu Ile His 20 25 30 Ala Arg Phe Val Met ThrAla Ala Ser Cys Phe Gln Ser Gln Asn Pro 35 40 45 Gly Val Ser Thr Val ValLeu Gly Ala Tyr Asp Leu Arg Arg Arg Glu 50 55 60 Arg Gln Ser Arg Gln ThrPhe Ser Ile Ser Ser Met Ser Glu Asn Gly 65 70 75 80 Tyr Asp Pro Gln GlnAsn Leu Asn Asp Leu Met Leu Leu Gln Leu Asp 85 90 95 Arg Glu Ala Asn LeuThr Ser Ser Val Thr Ile Leu Pro Leu Pro Leu 100 105 110 Gln Asn Ala ThrVal Glu Ala Gly Thr Arg Cys Gln Val Ala Gly Trp 115 120 125 Gly Ser GlnArg Ser Gly Gly Arg Leu Ser Arg Phe Pro Arg Phe Val 130 135 140 Asn ValThr Val Thr Pro Glu Asp Gln Cys Arg Pro Asn Asn Val Cys 145 150 155 160Thr Gly Val Leu Thr Arg Arg Gly Gly Ile Cys Asn Gly Asp Gly Gly 165 170175 Thr Pro Leu Val Cys Glu Gly Leu Ala His Gly Val Ala Ser Phe Ser 180185 190 Leu Gly Pro Cys Gly Arg Gly Pro Asp Phe Phe Thr Arg Val Ala Leu195 200 205 Phe Arg Asp Trp Ile Asp Gly Val Leu Asn Asn Pro Gly Pro GlyPro 210 215 220 Ala 225 224 amino acids amino acid single linear None 33Ile Ile Asp Gly Ala Pro Cys Ala Arg Gly Ser His Pro Trp Gln Val 1 5 1015 Ala Leu Leu Ser Gly Asn Gln Leu His Cys Gly Gly Val Leu Val Asn 20 2530 Glu Arg Trp Val Leu Thr Ala Ala His Cys Lys Met Asn Glu Tyr Thr 35 4045 Val His Leu Gly Ser Asp Thr Leu Gly Asp Arg Arg Ala Gln Arg Ile 50 5560 Lys Ala Ser Lys Ser Phe Arg His Pro Gly Tyr Ser Thr Gln Thr His 65 7075 80 Val Asn Asp Leu Met Leu Val Lys Leu Asn Ser Gln Ala Arg Leu Ser 8590 95 Ser Met Val Lys Lys Val Arg Leu Pro Ser Arg Cys Glu Pro Pro Gly100 105 110 Thr Thr Cys Thr Val Ser Gly Trp Gly Thr Thr Thr Ser Pro AspVal 115 120 125 Thr Phe Pro Ser Asp Leu Met Cys Val Asp Val Lys Leu IleSer Pro 130 135 140 Gln Asp Cys Thr Lys Val Tyr Lys Asp Leu Leu Glu AsnSer Met Leu 145 150 155 160 Cys Ala Gly Ile Pro Asp Ser Lys Lys Asn AlaCys Asn Gly Asp Ser 165 170 175 Gly Gly Pro Leu Val Cys Arg Gly Thr LeuGln Gly Leu Val Ser Trp 180 185 190 Gly Thr Phe Pro Cys Gly Gln Pro AsnAsp Pro Gly Val Tyr Thr Gln 195 200 205 Val Cys Lys Phe Thr Lys Trp IleAsn Asp Thr Met Lys Lys His Arg 210 215 220 224 amino acids amino acidsingle linear None 34 Ile Val Gly Gly Tyr Ile Cys Glu Glu Asn Ser ValPro Tyr Gln Val 1 5 10 15 Ser Leu Asn Ser Gly Tyr His Phe Cys Gly GlySer Leu Ile Ser Glu 20 25 30 Gln Trp Val Val Ser Ala Gly His Cys Tyr LysSer Arg Ile Gln Val 35 40 45 Arg Leu Gly Glu His Asn Ile Glu Val Leu GluGly Asn Glu Gln Phe 50 55 60 Ile Asn Ala Ala Lys Ile Ile Arg His Pro LysTyr Asn Ser Arg Thr 65 70 75 80 Leu Asp Asn Asp Ile Leu Leu Ile Lys LeuSer Ser Pro Ala Val Ile 85 90 95 Asn Ser Arg Val Ser Ala Ile Ser Leu ProThr Ala Pro Pro Ala Ala 100 105 110 Gly Thr Glu Ser Leu Ile Ser Gly TrpGly Asn Thr Leu Ser Ser Gly 115 120 125 Ala Asp Tyr Pro Asp Glu Leu GlnCys Leu Asp Ala Pro Val Leu Ser 130 135 140 Gln Ala Glu Cys Glu Ala SerTyr Pro Gly Lys Ile Thr Asn Asn Met 145 150 155 160 Phe Cys Val Gly PheLeu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp 165 170 175 Ser Gly Gly ProVal Val Ser Asn Gly Glu Leu Gln Gly Ile Val Ser 180 185 190 Trp Gly TyrGly Cys Ala Gln Lys Asn Arg Pro Gly Val Tyr Thr Lys 195 200 205 Val TyrAsn Tyr Val Asp Trp Ile Lys Asp Thr Ile Ala Ala Asn Ser 210 215 220 224amino acids amino acid single linear None 35 Ile Val Gly Gly Tyr Thr CysGlu Glu Asn Ser Leu Pro Tyr Gln Val 1 5 10 15 Ser Leu Asn Ser Gly SerHis Phe Cys Gly Gly Ser Leu Ile Ser Glu 20 25 30 Gln Trp Val Val Ser AlaAla His Cys Tyr Lys Thr Arg Ile Gln Val 35 40 45 Arg Leu Gly Glu His AsnIle Lys Val Leu Glu Gly Asn Glu Gln Phe 50 55 60 Ile Asn Ala Ala Lys IleIle Arg His Pro Lys Tyr Asn Arg Asp Thr 65 70 75 80 Leu Asp Asn Asp IleMet Leu Ile Lys Leu Ser Ser Pro Ala Val Ile 85 90 95 Asn Ala Arg Val SerThr Ile Ser Leu Pro Thr Ala Pro Pro Ala Ala 100 105 110 Gly Thr Glu CysLeu Ile Ser Gly Trp Gly Asn Thr Leu Ser Phe Gly 115 120 125 Ala Asp TyrPro Asp Glu Leu Lys Cys Leu Asp Ala Pro Val Leu Thr 130 135 140 Gln AlaGlu Cys Lys Ala Ser Tyr Pro Gly Lys Ile Thr Asn Ser Met 145 150 155 160Phe Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser Cys Gln Arg Asp 165 170175 Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu Gln Gly Val Val Ser 180185 190 Trp Gly His Gly Cys Ala Trp Lys Asn Arg Pro Gly Val Tyr Thr Lys195 200 205 Val Tyr Asn Tyr Val Asp Trp Ile Lys Asp Thr Ile Ala Ala AsnSer 210 215 220 224 amino acids amino acid single linear None 36 Ile ValGly Gly Tyr Asn Cys Glu Glu Asn Ser Val Pro Tyr Gln Val 1 5 10 15 SerLeu Asn Ser Gly Tyr His Phe Cys Gly Gly Ser Leu Ile Asn Glu 20 25 30 GlnTrp Val Val Ser Ala Gly His Cys Tyr Lys Ser Arg Ile Gln Val 35 40 45 ArgLeu Gly Glu His Asn Ile Glu Val Leu Glu Gly Asn Glu Gln Phe 50 55 60 IleAsn Ala Ala Lys Ile Ile Arg His Pro Gln Tyr Asp Arg Lys Thr 65 70 75 80Leu Asn Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Arg Ala Val Ile 85 90 95Asn Ala Arg Val Ser Thr Ile Ser Leu Pro Thr Ala Pro Pro Ala Thr 100 105110 Gly Thr Lys Cys Leu Ile Ser Gly Trp Gly Asn Thr Ala Ser Ser Gly 115120 125 Ala Asp Tyr Pro Asp Glu Leu Gln Cys Leu Asp Ala Pro Val Leu Ser130 135 140 Gln Ala Lys Cys Glu Ala Ser Tyr Pro Gly Lys Ile Thr Ser AsnMet 145 150 155 160 Phe Cys Val Gly Phe Leu Glu Gly Gly Lys Asp Ser CysGln Gly Asp 165 170 175 Ser Gly Gly Pro Val Val Cys Asn Gly Gln Leu GlnGly Val Val Ser 180 185 190 Trp Gly Asp Gly Cys Ala Gln Lys Asn Lys ProGly Val Tyr Thr Lys 195 200 205 Val Tyr Asn Tyr Val Lys Trp Ile Lys AsnThr Ile Ala Ala Asn Ser 210 215 220 237 amino acids amino acid singlelinear None 37 Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro TrpGln Val 1 5 10 15 Ala Val Tyr Ser His Gly Trp Ala His Cys Gly Gly ValLeu Val His 20 25 30 Pro Gln Trp Val Leu Thr Ala Ala His Cys Leu Lys LysAsn Ser Gln 35 40 45 Val Trp Leu Gly Arg His Asn Leu Phe Glu Pro Glu AspThr Gly Gln 50 55 60 Arg Val Pro Val Ser His Ser Phe Pro His Pro Leu TyrAsn Met Ser 65 70 75 80 Leu Leu Lys His Gln Ser Leu Arg Pro Asp Glu AspSer Ser His Asp 85 90 95 Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Lys IleThr Asp Val Val 100 105 110 Lys Val Leu Gly Leu Pro Thr Gln Glu Pro AlaLeu Gly Thr Thr Cys 115 120 125 Tyr Ala Ser Gly Trp Gly Ser Ile Glu ProGlu Glu Phe Leu Arg Pro 130 135 140 Arg Ser Leu Gln Cys Val Ser Leu HisLeu Leu Ser Asn Asp Met Cys 145 150 155 160 Ala Arg Ala Tyr Ser Glu LysVal Thr Glu Phe Met Leu Cys Ala Gly 165 170 175 Leu Trp Thr Gly Gly LysAsp Thr Cys Gly Gly Asp Ser Gly Gly Pro 180 185 190 Leu Val Cys Asn GlyVal Leu Gln Gly Ile Thr Ser Trp Gly Pro Glu 195 200 205 Pro Cys Ala LeuPro Glu Lys Pro Ala Val Tyr Thr Lys Val Val His 210 215 220 Tyr Arg LysTrp Ile Lys Asp Thr Ile Ala Ala Asn Pro 225 230 235 237 amino acidsamino acid single linear None 38 Ile Val Gly Gly Trp Glu Cys Glu Lys HisSer Gln Pro Trp Gln Val 1 5 10 15 Leu Val Ala Ser Arg Gly Arg Ala ValCys Gly Gly Val Leu Val His 20 25 30 Pro Gln Trp Val Leu Thr Ala Ala HisCys Ile Arg Asn Lys Ser Val 35 40 45 Ile Leu Leu Gly Arg His Ser Leu PheHis Pro Glu Asp Thr Gly Gln 50 55 60 Val Phe Gln Val Ser His Ser Phe ProHis Pro Leu Tyr Asp Met Ser 65 70 75 80 Leu Leu Lys Asn Arg Phe Leu ArgPro Gly Asp Asp Ser Ser His Asp 85 90 95 Leu Met Leu Leu Arg Leu Ser GluPro Ala Glu Leu Thr Asp Ala Val 100 105 110 Lys Val Met Asp Leu Pro ThrGln Glu Pro Ala Leu Gly Thr Thr Cys 115 120 125 Tyr Ala Ser Gly Trp GlySer Ile Glu Pro Glu Glu Phe Leu Thr Pro 130 135 140 Lys Lys Leu Gln CysVal Asp Leu His Val Ile Ser Asn Asp Val Cys 145 150 155 160 Ala Gln ValHis Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala Gly 165 170 175 Arg TrpThr Gly Gly Lys Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro 180 185 190 LeuVal Cys Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly Ser Glu 195 200 205Pro Cys Ala Leu Pro Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His 210 215220 Tyr Arg Lys Trp Ile Lys Asp Thr Ile Val Ala Asn Pro 225 230 235 238amino acids amino acid single linear None 39 Ile Val Gly Gly Trp Glu CysGlu Gln His Ser Gln Pro Trp Gln Ala 1 5 10 15 Ala Leu Tyr His Phe SerThr Phe Gln Cys Gly Gly Ile Leu Val His 20 25 30 Arg Gln Trp Val Leu ThrAla Ala His Cys Ile Ser Asp Asn Tyr Gln 35 40 45 Leu Trp Leu Gly Arg HisAsn Leu Phe Asp Asp Glu Asn Thr Ala Gln 50 55 60 Phe Val His Val Ser GluSer Phe Pro His Pro Gly Phe Asn Met Ser 65 70 75 80 Leu Leu Glu Asn HisThr Arg Gln Ala Asp Glu Asp Tyr Ser His Asp 85 90 95 Leu Met Leu Leu ArgLeu Thr Glu Pro Ala Asp Thr Ile Thr Asp Ala 100 105 110 Val Lys Val ValGlu Leu Pro Thr Gln Glu Pro Glu Val Gly Ser Thr 115 120 125 Cys Leu AlaSer Gly Trp Gly Ser Ile Glu Pro Glu Asn Phe Ser Phe 130 135 140 Pro AspAsp Leu Gln Cys Val Asp Leu Lys Ile Leu Pro Asn Asp Glu 145 150 155 160Cys Glu Lys Ala His Val Gln Lys Val Thr Asp Phe Met Leu Cys Val 165 170175 Gly His Leu Glu Gly Gly Lys Asp Thr Cys Val Gly Asp Ser Gly Gly 180185 190 Pro Leu Met Cys Asp Gly Val Leu Gln Gly Val Thr Ser Trp Gly Tyr195 200 205 Val Pro Cys Gly Thr Pro Asn Lys Pro Ser Val Ala Val Arg ValLeu 210 215 220 Ser Tyr Val Lys Trp Ile Glu Asp Thr Ile Ala Glu Asn Ser225 230 235 227 amino acids amino acid single linear None 40 Ile Ile GlyGly His Glu Ala Lys Pro His Ser Arg Pro Tyr Met Ala 1 5 10 15 Tyr LeuMet Ile Trp Asp Gln Lys Ser Leu Lys Arg Cys Gly Gly Phe 20 25 30 Leu IleGln Asp Asp Phe Val Leu Thr Ala Ala His Cys Trp Gly Ser 35 40 45 Ser IleAsn Val Thr Leu Gly Ala His Asn Ile Lys Glu Gln Glu Pro 50 55 60 Thr GlnGln Phe Ile Pro Val Lys Arg Ala Ile Pro His Pro Ala Tyr 65 70 75 80 AsnPro Lys Asn Phe Ser Asn Asp Ile Met Leu Leu Gln Leu Glu Arg 85 90 95 LysAla Lys Arg Thr Arg Ala Val Gln Pro Leu Arg Leu Pro Ser Asn 100 105 110Lys Ala Gln Val Lys Pro Gly Gln Thr Cys Ser Val Ala Gly Trp Gly 115 120125 Gln Thr Ala Pro Leu Gly Lys His Ser His Thr Leu Gln Glu Val Lys 130135 140 Met Thr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp Leu Arg His Tyr145 150 155 160 Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro Glu IleLys Lys 165 170 175 Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val CysAsn Lys Val 180 185 190 Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn GlyMet Pro Pro Arg 195 200 205 Ala Cys Thr Lys Val Ser Ser Phe Val His TrpIle Lys Lys Thr Met 210 215 220 Lys Arg Tyr 225 226 amino acids aminoacid single linear None 41 Ile Ile Gly Gly His Glu Ala Lys Pro His SerArg Pro Tyr Met Ala 1 5 10 15 Phe Val Gln Phe Leu Gln Glu Lys Ser ArgLys Arg Cys Gly Gly Ile 20 25 30 Leu Val Arg Lys Asp Phe Val Leu Thr AlaAla His Cys Gln Gly Ser 35 40 45 Ser Ile Asn Val Thr Leu Gly Ala His AsnIle Lys Glu Gln Glu Arg 50 55 60 Thr Gln Gln Phe Ile Pro Val Lys Arg ProIle Pro His Pro Ala Tyr 65 70 75 80 Asn Pro Lys Asn Phe Ser Asn Asp IleMet Leu Leu Gln Leu Glu Arg 85 90 95 Lys Ala Lys Trp Thr Thr Ala Val ArgPro Leu Arg Leu Pro Ser Ser 100 105 110 Lys Ala Gln Val Lys Pro Gly GlnLeu Cys Ser Val Ala Gly Trp Gly 115 120 125 Tyr Val Ser Met Ser Thr LeuAla Thr Thr Leu Gln Glu Val Leu Leu 130 135 140 Thr Val Gln Lys Asp CysGln Cys Glu Arg Leu Phe His Gly Asn Tyr 145 150 155 160 Ser Arg Ala ThrGlu Ile Cys Val Gly Asp Pro Lys Lys Thr Gln Thr 165 170 175 Gly Phe LysGly Asp Ser Gly Gly Pro Leu Val Cys Lys Asp Val Ala 180 185 190 Gln GlyIle Leu Ser Tyr Gly Asn Lys Lys Gly Thr Pro Pro Gly Val 195 200 205 TyrIle Lys Val Ser His Phe Leu Pro Trp Ile Lys Arg Thr Met Lys 210 215 220Arg Leu 225 235 amino acids amino acid single linear None 42 Ile Ile GlyGly Arg Glu Ser Arg Pro His Ser Arg Pro Tyr Met Ala 1 5 10 15 Tyr LeuGln Ile Gln Ser Pro Ala Gly Gln Ser Arg Cys Gly Gly Phe 20 25 30 Leu ValArg Glu Asp Phe Val Leu Thr Ala Ala His Cys Trp Gly Ser 35 40 45 Asn IleAsn Val Thr Leu Gly Ala His Asn Ile Gln Arg Arg Glu Asn 50 55 60 Thr GlnGln His Ile Thr Ala Arg Arg Ala Ile Arg His Pro Gln Tyr 65 70 75 80 AsnGln Arg Thr Ile Gln Asn Asp Ile Met Leu Leu Gln Leu Ser Arg 85 90 95 ArgVal Arg Arg Asn Arg Asn Val Asn Pro Val Ala Leu Pro Arg Ala 100 105 110Gln Glu Gly Leu Arg Pro Gly Thr Leu Cys Thr Val Ala Gly Trp Gly 115 120125 Arg Val Ser Met Arg Arg Gly Thr Asp Thr Leu Arg Glu Val Gln Leu 130135 140 Arg Val Gln Arg Asp Arg Gln Cys Leu Arg Ile Phe Gly Ser Tyr Asp145 150 155 160 Pro Arg Arg Gln Ile Cys Val Gly Asp Arg Arg Glu Arg LysAla Ala 165 170 175 Phe Lys Gly Asp Ser Gly Gly Pro Leu Leu Cys Asn AsnVal Ala His 180 185 190 Gly Ile Val Ser Tyr Gly Lys Ser Ser Gly Val ProPro Glu Val Phe 195 200 205 Thr Arg Val Ser Ser Phe Leu Pro Trp Ile ArgThr Thr Met Arg Ser 210 215 220 Phe Lys Leu Leu Asp Gln Met Glu Thr ProLeu 225 230 235 226 amino acids amino acid single linear None 43 Ile IleGly Gly Thr Glu Cys Lys Pro His Ser Arg Pro Tyr Met Ala 1 5 10 15 TyrLeu Glu Ile Val Thr Ser Asn Gly Pro Ser Lys Phe Cys Gly Gly 20 25 30 PheLeu Ile Arg Arg Asn Phe Val Leu Thr Ala Ala His Cys Ala Gly 35 40 45 ArgSer Ile Thr Val Thr Leu Gly Ala His Asn Ile Thr Glu Glu Glu 50 55 60 AspThr Trp Gln Lys Leu Glu Val Ile Lys Gln Phe Arg His Pro Lys 65 70 75 80Tyr Asn Thr Ser Thr Leu His His Asp Ile Met Leu Leu Lys Leu Lys 85 90 95Glu Lys Ala Ser Leu Thr Leu Ala Val Gly Thr Leu Pro Phe Pro Ser 100 105110 Gln Phe Asn Phe Val Pro Pro Gly Arg Met Cys Arg Val Ala Gly Trp 115120 125 Gly Arg Thr Gly Val Leu Lys Pro Gly Ser Asp Thr Leu Gln Glu Val130 135 140 Lys Leu Arg Leu Met Asp Pro Gln Ala Cys Ser His Phe Arg AspPhe 145 150 155 160 Asp His Asn Leu Gln Leu Cys Val Gly Asn Pro Arg LysThr Lys Ser 165 170 175 Ala Phe Lys Gly Asp Ser Gly Gly Pro Leu Leu CysAla Gly Val Ala 180 185 190 Gln Gly Ile Val Ser Tyr Gly Arg Ser Asp AlaLys Pro Pro Ala Val 195 200 205 Phe Thr Arg Ile Ser His Tyr Arg Pro TrpIle Asn Gln Ile Leu Gln 210 215 220 Ala Asn 225 228 amino acids aminoacid single linear None 44 Met Leu Gly Gly Arg Glu Ala Glu Ala His AlaArg Pro Tyr Met Ala 1 5 10 15 Ser Val Gln Leu Asn Gly Ala His Leu CysAla Gly Val Leu Val Ala 20 25 30 Glu Arg Trp Val Leu Ser Ala Ala His CysLeu Glu Asp Ala Ala Asp 35 40 45 Gly Lys Val Gln Val Leu Leu Gly Ala HisSer Leu Ser Gln Pro Glu 50 55 60 Pro Ser Lys Arg Leu Tyr Asp Val Leu ArgAla Val Pro His Pro Asp 65 70 75 80 Ser Gln Pro Asp Thr Ile Asp His AspLeu Leu Leu Leu Gln Leu Ser 85 90 95 Glu Lys Ala Thr Leu Gly Pro Ala ValArg Pro Leu Pro Trp Gln Arg 100 105 110 Val Asp Arg Asp Val Ala Pro GlyThr Leu Cys Asp Val Ala Gly Trp 115 120 125 Gly Ile Val Asn His Ala GlyArg Arg Pro Asp Ser Leu Gln His Val 130 135 140 Leu Leu Pro Val Leu AspArg Ala Thr Cys Asn Arg Arg Thr His His 145 150 155 160 Asp Gly Ala IleThr Glu Arg Leu Met Cys Ala Glu Ser Asn Arg Arg 165 170 175 Asp Ser CysLys Gly Asp Ser Gly Gly Pro Leu Val Cys Gly Gly Val 180 185 190 Leu GluGly Val Val Thr Ser Gly Ser Arg Val Cys Gly Asn Arg Lys 195 200 205 LysPro Gly Ile Tyr Thr Arg Val Ala Ser Tyr Ala Ala Trp Ile Asp 210 215 220Ser Val Leu Ala 225 232 amino acids amino acid single linear None 45 IleIle Gly Gly Arg Glu Val Ile Pro His Ser Arg Pro Tyr Met Ala 1 5 10 15Ser Leu Gln Arg Asn Gly Ser His Leu Cys Gly Gly Val Leu Val His 20 25 30Pro Lys Trp Val Leu Thr Ala Ala His Cys Leu Ala Gln Arg Met Ala 35 40 45Gln Leu Arg Leu Val Leu Gly Leu His Thr Leu Asp Ser Pro Gly Leu 50 55 60Thr Phe His Ile Lys Ala Ala Ile Gln His Pro Arg Tyr Lys Pro Val 65 70 7580 Pro Ala Leu Glu Asn Asp Leu Ala Leu Leu Gln Leu Asp Gly Lys Val 85 9095 Lys Pro Ser Arg Thr Ile Arg Pro Leu Ala Leu Pro Ser Lys Arg Gln 100105 110 Val Val Ala Ala Gly Thr Arg Cys Ser Met Ala Gly Trp Gly Leu Thr115 120 125 His Gln Gly Gly Arg Leu Ser Arg Val Leu Arg Glu Leu Asp LeuGln 130 135 140 Val Leu Asp Thr Arg Met Cys Asn Asn Ser Arg Phe Trp AsnGly Ser 145 150 155 160 Leu Ser Pro Ser Met Val Cys Leu Ala Ala Asp SerLys Asp Gln Ala 165 170 175 Pro Cys Lys Gly Asp Ser Gly Gly Pro Leu ValCys Gly Lys Gly Arg 180 185 190 Val Leu Ala Gly Val Leu Ser Phe Ser SerArg Val Cys Thr Asp Ile 195 200 205 Phe Lys Pro Pro Val Ala Thr Ala ValAla Pro Tyr Val Ser Trp Ile 210 215 220 Arg Lys Val Thr Gly Arg Ser Ala225 230 234 amino acids amino acid single linear None 46 Ile Ile Gly GlyAsn Glu Val Thr Pro His Ser Arg Pro Tyr Met Val 1 5 10 15 Leu Leu SerLeu Asp Arg Lys Thr Ile Cys Ala Gly Ala Leu Ile Ala 20 25 30 Lys Asp TrpVal Leu Thr Ala Ala His Cys Asn Leu Asn Lys Arg Ser 35 40 45 Gln Val IleLeu Gly Ala His Ser Ile Thr Arg Glu Glu Pro Thr Lys 50 55 60 Gln Ile MetLeu Val Lys Lys Glu Phe Pro Tyr Pro Cys Tyr Asp Pro 65 70 75 80 Ala ThrArg Glu Gly Asp Leu Lys Leu Leu Gln Leu Thr Glu Lys Ala 85 90 95 Lys IleAsn Lys Tyr Val Thr Ile Leu His Leu Pro Lys Lys Gly Asp 100 105 110 AspVal Lys Pro Gly Thr Met Cys Gln Val Ala Gly Trp Gly Arg Thr 115 120 125His Asn Ser Ala Ser Trp Ser Asp Thr Leu Arg Glu Val Asn Ile Thr 130 135140 Ile Ile Asp Arg Lys Val Cys Asn Asp Arg Asn His Tyr Asn Phe Asn 145150 155 160 Pro Val Ile Gly Met Asn Met Val Cys Ala Gly Ser Leu Arg GlyGly 165 170 175 Arg Asp Ser Cys Asn Gly Asp Ser Gly Ser Pro Leu Leu CysGlu Gly 180 185 190 Val Phe Arg Gly Val Thr Ser Phe Gly Leu Glu Asn LysCys Gly Asp 195 200 205 Pro Arg Gly Pro Gly Val Tyr Ile Leu Leu Ser LysLys His Leu Asn 210 215 220 Trp Ile Ile Met Thr Ile Lys Gly Ala Val 225230 251 amino acids amino acid single linear None 47 Ile Phe Asn Gly ArgPro Ala Gln Lys Gly Thr Thr Pro Trp Ile Ala 1 5 10 15 Met Leu Ser HisLeu Asn Gly Gln Pro Phe Cys Gly Gly Ser Leu Leu 20 25 30 Gly Ser Ser TrpIle Val Thr Ala Ala His Cys Leu His Gln Ser Leu 35 40 45 Asp Pro Lys AspPro Thr Leu Arg Asp Ser Asp Leu Leu Ser Pro Ser 50 55 60 Asp Phe Lys IleIle Leu Gly Lys His Trp Arg Leu Arg Ser Asp Glu 65 70 75 80 Asn Glu GlnHis Leu Gly Val Lys His Thr Thr Leu His Pro Lys Tyr 85 90 95 Asp Pro AsnThr Phe Glu Asn Asp Val Ala Leu Val Glu Leu Leu Glu 100 105 110 Ser ProVal Leu Asn Ala Phe Val Met Pro Ile Cys Leu Pro Glu Gly 115 120 125 ProGln Gln Glu Gly Ala Met Val Ile Val Ser Gly Trp Gly Lys Gln 130 135 140Phe Leu Gln Arg Phe Pro Glu Thr Leu Met Glu Ile Glu Ile Pro Ile 145 150155 160 Val Asp His Ser Thr Cys Gln Lys Ala Tyr Ala Pro Leu Lys Lys Lys165 170 175 Val Thr Arg Asp Met Ile Cys Ala Gly Glu Lys Glu Gly Gly LysAsp 180 185 190 Ala Cys Ser Gly Asp Ser Gly Gly Pro Met Val Thr Leu AsnArg Glu 195 200 205 Arg Gly Gln Trp Tyr Leu Val Gly Thr Val Ser Trp GlyAsp Asp Cys 210 215 220 Gly Lys Lys Asp Arg Tyr Gly Val Tyr Ser Tyr IleHis His Asn Lys 225 230 235 240 Asp Trp Ile Gln Arg Val Thr Gly Val ArgAsn 245 250 235 amino acids amino acid single linear None 48 Val Val GlyGly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp Gln Val 1 5 10 15 Val LeuAsn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile Val Asn 20 25 30 Glu LysTrp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly Val Lys 35 40 45 Ile ThrVal Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu His Thr 50 55 60 Glu GlnLys Arg Asn Val Ile Arg Ile Ile Pro His His Asn Tyr Asn 65 70 75 80 AlaAla Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu Leu Asp 85 90 95 GluPro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile Ala Asp 100 105 110Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr Val Ser 115 120125 Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val Leu Gln 130135 140 Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg Ser Thr145 150 155 160 Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe HisGlu Gly 165 170 175 Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro HisVal Thr Glu 180 185 190 Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile SerTrp Gly Glu Glu 195 200 205 Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr ThrLys Val Ser Arg Tyr 210 215 220 Val Asn Trp Ile Lys Glu Lys Thr Lys LeuThr 225 230 235 247 amino acids amino acid single linear None 49 Ile ValGly Gly Gln Glu Cys Lys Asp Gly Glu Cys Pro Trp Gln Ala 1 5 10 15 LeuLeu Ile Asn Glu Glu Asn Glu Gly Phe Cys Gly Gly Thr Ile Leu 20 25 30 SerGlu Phe Tyr Ile Leu Thr Ala Ala His Cys Leu Tyr Gln Ala Lys 35 40 45 ArgPhe Glu Gly Asp Arg Asn Thr Glu Gln Glu Glu Gly Gly Glu Ala 50 55 60 ValHis Glu Val Glu Val Val Ile Lys His Asn Arg Phe Thr Lys Glu 65 70 75 80Thr Tyr Asp Phe Asp Ile Ala Val Leu Arg Leu Lys Thr Pro Ile Thr 85 90 95Phe Arg Met Asn Val Ala Pro Ala Cys Leu Pro Glu Arg Asp Trp Ala 100 105110 Glu Ser Thr Leu Met Thr Gln Lys Thr Gly Ile Val Ser Gly Phe Gly 115120 125 Arg Thr His Glu Lys Gly Arg Gln Ser Thr Arg Leu Lys Met Leu Glu130 135 140 Val Pro Tyr Val Asp Arg Asn Ser Cys Lys Leu Ser Ser Ser PheIle 145 150 155 160 Ile Thr Gln Asn Met Phe Cys Ala Gly Tyr Asp Thr LysGln Glu Asp 165 170 175 Ala Cys Gln Gly Asp Ser Gly Gly Pro His Val ThrArg Phe Lys Asp 180 185 190 Thr Tyr Phe Val Thr Gly Ile Val Ser Trp GlyGlu Gly Cys Ala Arg 195 200 205 Lys Gly Lys Tyr Gly Ile Tyr Thr Lys ValThr Ala Phe Leu Lys Trp 210 215 220 Ile Asp Arg Ser Met Lys Thr Arg GlyLeu Pro Lys Ala Lys Ser His 225 230 235 240 Ala Pro Glu Val Ile Thr Ser245 254 amino acids amino acid single linear None 50 Ile Val Gly Gly LysVal Cys Pro Lys Gly Glu Cys Pro Trp Gln Val 1 5 10 15 Leu Leu Leu ValAsn Gly Ala Gln Leu Cys Gly Gly Thr Leu Ile Asn 20 25 30 Thr Ile Trp ValVal Ser Ala Ala His Cys Phe Asp Lys Ile Lys Asn 35 40 45 Trp Arg Asn LeuIle Ala Val Leu Gly Glu His Asp Leu Ser Glu His 50 55 60 Asp Gly Asp GluGln Ser Arg Arg Val Ala Gln Val Ile Ile Pro Ser 65 70 75 80 Thr Tyr ValPro Gly Thr Thr Asn His Asp Ile Ala Leu Leu Arg Leu 85 90 95 His Gln ProVal Val Leu Thr Asp His Val Val Pro Leu Cys Leu Pro 100 105 110 Glu ArgThr Phe Ser Glu Arg Thr Leu Ala Phe Val Arg Phe Ser Leu 115 120 125 ValSer Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala Thr Ala Leu Glu 130 135 140Leu Met Val Leu Asn Val Pro Arg Leu Met Thr Gln Asp Cys Leu Gln 145 150155 160 Gln Ser Arg Lys Val Gly Asp Ser Pro Asn Ile Thr Glu Tyr Met Phe165 170 175 Cys Ala Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys Lys Gly AspSer 180 185 190 Gly Gly Pro His Ala Thr His Tyr Arg Gly Thr Trp Tyr LeuThr Gly 195 200 205 Ile Val Ser Trp Gly Gln Gly Cys Ala Thr Val Gly HisPhe Gly Val 210 215 220 Tyr Thr Arg Val Ser Gln Tyr Ile Glu Trp Leu GlnLys Leu Met Arg 225 230 235 240 Ser Glu Pro Arg Pro Gly Val Leu Leu ArgAla Pro Phe Pro 245 250 250 amino acids amino acid single linear None 51Leu Ile Asp Gly Lys Met Thr Arg Arg Gly Asp Ser Pro Trp Gln Val 1 5 1015 Val Leu Leu Asp Ser Lys Lys Lys Leu Ala Cys Gly Ala Val Leu Ile 20 2530 His Pro Ser Trp Val Leu Thr Ala Ala His Cys Met Asp Glu Ser Lys 35 4045 Lys Leu Leu Val Arg Leu Gly Glu Tyr Asp Leu Arg Arg Trp Glu Lys 50 5560 Trp Glu Leu Asp Leu Asp Ile Lys Glu Val Phe Val His Pro Asn Tyr 65 7075 80 Ser Lys Ser Thr Thr Asp Asn Asp Ile Ala Leu Leu His Leu Ala Gln 8590 95 Pro Ala Thr Leu Ser Gln Thr Ile Val Pro Ile Cys Leu Pro Asp Ser100 105 110 Gly Leu Ala Glu Arg Glu Leu Asn Gln Ala Gly Gln Glu Thr LeuVal 115 120 125 Thr Gly Trp Gly Tyr His Ser Ser Arg Glu Lys Glu Ala LysArg Asn 130 135 140 Arg Thr Phe Val Leu Asn Phe Ile Lys Ile Pro Val ValPro His Asn 145 150 155 160 Glu Cys Ser Glu Val Met Ser Asn Met Val SerGlu Asn Met Leu Cys 165 170 175 Ala Gly Ile Leu Gly Asp Arg Gln Asp AlaCys Glu Gly Asp Ser Gly 180 185 190 Gly Pro Met Val Ala Ser Phe His GlyThr Trp Phe Leu Val Gly Leu 195 200 205 Val Ser Trp Gly Glu Gly Cys GlyLeu Leu His Asn Tyr Gly Val Tyr 210 215 220 Thr Lys Val Ser Arg Tyr LeuAsp Trp Ile His Gly His Ile Arg Asp 225 230 235 240 Lys Glu Ala Pro GlnLys Ser Trp Ala Pro 245 250 259 amino acids amino acid single linearNone 52 Ile Val Glu Gly Ser Asp Ala Glu Ile Gly Met Ser Pro Trp Gln Val1 5 10 15 Met Leu Phe Arg Lys Ser Pro Gln Glu Leu Leu Cys Gly Ala SerLeu 20 25 30 Ile Ser Asp Arg Trp Val Leu Thr Ala Ala His Cys Leu Leu TyrPro 35 40 45 Pro Trp Asp Lys Asn Phe Thr Glu Asn Asp Leu Leu Val Arg IleGly 50 55 60 Lys His Ser Arg Thr Arg Tyr Glu Arg Asn Ile Glu Lys Ile SerMet 65 70 75 80 Leu Glu Lys Ile Tyr Ile His Pro Arg Tyr Asn Trp Arg GluAsn Leu 85 90 95 Asp Arg Asp Ile Ala Leu Met Lys Leu Lys Lys Pro Val AlaPhe Ser 100 105 110 Asp Tyr Ile His Pro Val Cys Leu Pro Asp Arg Glu ThrAla Ala Ser 115 120 125 Leu Leu Gln Ala Gly Tyr Lys Gly Arg Val Thr GlyTrp Gly Asn Leu 130 135 140 Lys Glu Thr Trp Thr Ala Asn Val Gly Lys GlyGln Pro Ser Val Leu 145 150 155 160 Gln Val Val Asn Leu Pro Ile Val GluArg Pro Val Cys Lys Asp Ser 165 170 175 Thr Arg Ile Arg Ile Thr Asp AsnMet Phe Cys Ala Gly Tyr Lys Pro 180 185 190 Asp Glu Gly Lys Arg Gly AspAla Cys Glu Gly Asp Ser Gly Gly Pro 195 200 205 Phe Val Met Lys Ser ProPhe Asn Asn Arg Trp Tyr Gln Met Gly Ile 210 215 220 Val Ser Trp Gly GluGly Cys Asp Arg Asp Gly Lys Tyr Gly Phe Tyr 225 230 235 240 Thr His ValPhe Arg Leu Lys Lys Trp Ile Gln Lys Val Ile Asp Gln 245 250 255 Phe GlyGlu 230 amino acids amino acid single linear None 53 Val Val Gly Gly CysVal Ala His Pro His Ser Trp Pro Trp Gln Val 1 5 10 15 Ser Leu Arg ThrArg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile 20 25 30 Ser Pro Glu TrpVal Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro 35 40 45 Arg Pro Ser SerTyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn 50 55 60 Leu Glu Pro HisVal Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu 65 70 75 80 Pro Thr ArgLys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val 85 90 95 Ile Thr AspLys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val 100 105 110 Val AlaAsp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln 115 120 125 GlyThr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile 130 135 140Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln 145 150155 160 Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys165 170 175 Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp LysTyr 180 185 190 Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala ArgPro Asn 195 200 205 Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val ThrTrp Ile Glu 210 215 220 Gly Val Met Arg Asn Asn 225 230 221 amino acidsamino acid single linear None 54 Ile Val Gly Gly Cys Val Ala His Pro HisSer Trp Pro Trp Gln Val 1 5 10 15 Ser Leu Arg Thr Arg Phe Gly Lys HisPhe Cys Gly Gly Thr Leu Ile 20 25 30 Ser Pro Glu Trp Val Leu Thr Ala AlaHis Cys Leu Lys Lys Ser Ser 35 40 45 Arg Pro Ser Ser Tyr Lys Val Ile LeuGly Ala His Gln Glu Val Asn 50 55 60 Leu Glu Ser His Val Gln Glu Ile GluVal Ser Arg Leu Phe Leu Glu 65 70 75 80 Pro Thr Gln Ala Asp Ile Ala LeuLeu Lys Leu Ser Arg Pro Ala Val 85 90 95 Ile Thr Asp Lys Val Met Pro AlaCys Leu Pro Ser Pro Asp Tyr Met 100 105 110 Val Thr Ala Arg Thr Glu CysTyr Ile Thr Gly Trp Gly Glu Thr Gln 115 120 125 Gly Thr Phe Gly Thr GlyLeu Leu Lys Glu Ala Gln Leu Leu Val Ile 130 135 140 Glu Asn Glu Val CysAsn His Tyr Lys Tyr Ile Cys Ala Glu His Leu 145 150 155 160 Ala Arg GlyThr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val 165 170 175 Cys PheGlu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly 180 185 190 LeuGly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Ala Arg Val Ser 195 200 205Arg Phe Val Thr Trp Ile Glu Gly Met Met Arg Asn Asn 210 215 220 228amino acids amino acid single linear None 55 Val Val Gly Gly His Pro GlyAsn Ser Pro Trp Thr Val Ser Leu Arg 1 5 10 15 Asn Arg Gln Gly Gln HisPhe Cys Gly Gly Ser Leu Val Lys Glu Gln 20 25 30 Trp Ile Leu Thr Ala ArgGln Cys Phe Ser Ser Cys His Met Pro Leu 35 40 45 Thr Gly Tyr Glu Val TrpLeu Gly Thr Leu Phe Gln Asn Pro Gln His 50 55 60 Gly Glu Pro Ser Leu GlnArg Val Pro Val Ala Lys Met Val Cys Gly 65 70 75 80 Pro Ser Gly Ser GlnLeu Val Leu Leu Lys Leu Glu Arg Ser Val Thr 85 90 95 Leu Asn Gln Arg ValAla Leu Ile Cys Leu Pro Pro Glu Trp Tyr Val 100 105 110 Val Pro Pro GlyThr Lys Cys Glu Ile Ala Gly Trp Gly Glu Thr Lys 115 120 125 Gly Thr GlyAsn Asp Thr Val Leu Asn Val Ala Phe Leu Asn Val Ile 130 135 140 Ser AsnGln Glu Cys Asn Ile Lys His Arg Gly Arg Val Arg Glu Ser 145 150 155 160Glu Met Cys Thr Glu Gly Leu Leu Ala Pro Val Gly Ala Cys Glu Gly 165 170175 Asp Tyr Gly Gly Pro Leu Ala Cys Phe Thr His Asn Cys Trp Val Leu 180185 190 Glu Gly Ile Ile Ile Pro Asn Arg Val Cys Ala Arg Ser Arg Trp Pro195 200 205 Ala Val Phe Thr Arg Val Ser Val Phe Val Asp Trp Ile His LysVal 210 215 220 Met Arg Leu Gly 225 234 amino acids amino acid singlelinear None 56 Val Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp MetVal Ser 1 5 10 15 Leu Arg Tyr Arg Asn Lys His Ile Cys Gly Gly Ser LeuIle Lys Glu 20 25 30 Ser Trp Val Leu Thr Ala Arg Gln Cys Phe Pro Ser ArgAsp Leu Lys 35 40 45 Asp Tyr Glu Ala Trp Leu Gly Ile His Asp Val His GlyArg Gly Asp 50 55 60 Glu Lys Cys Lys Gln Val Leu Asn Val Ser Gln Leu ValTyr Gly Pro 65 70 75 80 Glu Gly Ser Asp Leu Val Leu Met Lys Leu Ala ArgPro Ala Val Leu 85 90 95 Asp Asp Phe Val Ser Thr Ile Asp Leu Pro Asn TyrGly Cys Thr Ile 100 105 110 Pro Glu Lys Thr Ser Cys Ser Val Tyr Gly TrpGly Tyr Thr Gly Leu 115 120 125 Ile Asn Tyr Asp Gly Leu Leu Arg Val AlaHis Leu Tyr Ile Met Gly 130 135 140 Asn Glu Lys Cys Ser Gln His His ArgGly Lys Val Thr Leu Asn Glu 145 150 155 160 Ser Glu Ile Cys Ala Gly AlaGlu Lys Ile Gly Ser Gly Pro Cys Glu 165 170 175 Gly Asp Tyr Gly Gly ProLeu Val Cys Glu Gln His Lys Met Arg Met 180 185 190 Val Leu Gly Val IleVal Pro Gly Arg Gly Cys Ala Ile Pro Asn Arg 195 200 205 Pro Gly Ile PheVal Arg Val Ala Tyr Tyr Ala Lys Trp Ile His Lys 210 215 220 Ile Ile LeuThr Tyr Lys Val Pro Gln Ser 225 230 242 amino acids amino acid singlelinear None 57 Val Val Asn Gly Glu Asp Ala Val Pro Tyr Ser Trp Pro TrpGln Val 1 5 10 15 Ser Leu Gln Tyr Glu Lys Ser Gly Ser Phe Tyr His ThrCys Gly Gly 20 25 30 Ser Leu Ile Ala Pro Asp Trp Val Val Thr Ala Gly HisCys Ile Ser 35 40 45 Ser Ser Arg Thr Tyr Gln Val Val Leu Gly Glu Tyr AspArg Ala Val 50 55 60 Lys Glu Gly Pro Glu Gln Val Ile Pro Ile Asn Ser GlyAsp Leu Phe 65 70 75 80 Val His Pro Leu Trp Asn Arg Ser Cys Val Ala CysGly Asn Asp Ile 85 90 95 Ala Leu Ile Lys Leu Ser Arg Ser Ala Gln Leu GlyAsp Ala Val Gln 100 105 110 Leu Ala Ser Leu Pro Pro Ala Gly Asp Ile LeuPro Asn Glu Thr Pro 115 120 125 Cys Tyr Ile Thr Gly Trp Gly Arg Leu TyrThr Asn Gly Pro Leu Pro 130 135 140 Asp Lys Leu Gln Glu Ala Leu Leu ProVal Val Asp Tyr Glu His Cys 145 150 155 160 Ser Arg Trp Asn Trp Trp GlySer Ser Val Lys Lys Thr Met Val Cys 165 170 175 Ala Gly Gly Asp Ile ArgSer Gly Cys Asn Gly Asp Ser Gly Gly Pro 180 185 190 Leu Asn Cys Pro ThrGlu Asp Gly Gly Trp Gln Val His Gly Val Thr 195 200 205 Ser Phe Val SerAla Phe Gly Cys Asn Thr Arg Arg Lys Pro Thr Val 210 215 220 Phe Thr ArgVal Ser Ala Phe Ile Asp Trp Ile Glu Glu Thr Ile Ala 225 230 235 240 SerHis 242 amino acids amino acid single linear None 58 Val Val His Gly GluAsp Ala Val Pro Tyr Ser Trp Pro Trp Gln Val 1 5 10 15 Ser Leu Gln TyrGlu Lys Ser Gly Ser Phe Tyr His Thr Cys Gly Gly 20 25 30 Ser Leu Ile AlaPro Asp Trp Val Val Thr Ala Gly His Cys Ile Ser 35 40 45 Arg Asp Leu ThrTyr Gln Val Val Leu Gly Glu Tyr Asn Leu Ala Val 50 55 60 Lys Glu Gly ProGlu Gln Val Ile Pro Ile Asn Ser Glu Glu Leu Phe 65 70 75 80 Val His ProLeu Trp Asn Arg Ser Cys Val Ala Cys Gly Asn Asp Ile 85 90 95 Ala Leu IleLys Leu Ser Arg Ser Ala Gln Leu Gly Asp Ala Val Gln 100 105 110 Leu AlaSer Leu Pro Pro Ala Gly Asp Ile Leu Pro Asn Lys Thr Pro 115 120 125 CysTyr Ile Thr Gly Trp Gly Arg Leu Tyr Thr Asn Gly Pro Leu Pro 130 135 140Asp Lys Leu Gln Gln Ala Arg Leu Pro Val Val Asp Tyr Lys His Cys 145 150155 160 Ser Arg Trp Asn Trp Trp Gly Ser Thr Val Lys Lys Thr Met Val Cys165 170 175 Ala Gly Gly Tyr Ile Arg Ser Gly Cys Asn Gly Asp Ser Gly GlyPro 180 185 190 Leu Asn Cys Pro Thr Glu Asp Gly Gly Trp Gln Val His GlyVal Thr 195 200 205 Ser Phe Val Ser Gly Phe Gly Cys Asn Phe Ile Trp LysPro Thr Val 210 215 220 Phe Thr Arg Val Ser Ala Phe Ile Asp Trp Ile GluGlu Thr Ile Ala 225 230 235 240 Ser His 241 amino acids amino acidsingle linear None 59 Val Val Gly Gly Glu Glu Ala Arg Pro Asn Ser TrpPro Trp Gln Val 1 5 10 15 Ser Leu Gln Tyr Ser Ser Asn Gly Lys Trp TyrHis Thr Cys Gly Gly 20 25 30 Ser Leu Ile Ala Asn Ser Trp Val Leu Thr AlaAla His Cys Ile Ser 35 40 45 Ser Ser Arg Thr Tyr Arg Val Gly Leu Gly ArgHis Asn Leu Tyr Val 50 55 60 Ala Glu Ser Gly Ser Leu Ala Val Ser Val SerLys Ile Val Val His 65 70 75 80 Lys Asp Trp Asn Ser Asn Gln Ile Ser LysGly Asn Asp Ile Ala Leu 85 90 95 Leu Lys Leu Ala Asn Pro Val Ser Leu ThrAsp Lys Ile Gln Leu Ala 100 105 110 Cys Leu Pro Pro Ala Gly Thr Ile LeuPro Asn Asn Tyr Pro Cys Tyr 115 120 125 Val Thr Gly Trp Gly Arg Leu GlnThr Asn Gly Ala Val Pro Asp Val 130 135 140 Leu Gln Gln Gly Arg Leu LeuVal Val Asp Tyr Ala Thr Cys Ser Ser 145 150 155 160 Ser Ala Trp Trp GlySer Ser Val Lys Thr Ser Met Ile Cys Ala Gly 165 170 175 Gly Asp Gly ValIle Ser Ser Cys Asn Gly Asp Ser Gly Gly Pro Leu 180 185 190 Asn Cys GlnAla Ser Asp Gly Arg Trp Gln Val His Gly Ile Val Ser 195 200 205 Phe GlySer Arg Leu Gly Cys Asn Tyr Tyr His Lys Pro Ser Val Phe 210 215 220 ThrArg Val Ser Asn Tyr Ile Asp Trp Ile Asn Ser Val Ile Ala Asn 225 230 235240 Asn 241 amino acids amino acid single linear None 60 Met Leu Gly GlyGlu Glu Ala Arg Pro Asn Ser Trp Pro Trp Gln Val 1 5 10 15 Ser Leu GlnTyr Ser Ser Asn Gly Gln Trp Tyr His Thr Cys Gly Gly 20 25 30 Ser Leu IleAla Asn Ser Trp Val Leu Thr Ala Ala His Cys Ile Ser 35 40 45 Ser Ser ArgIle Tyr Arg Val Met Leu Gly Gln His Asn Leu Tyr Val 50 55 60 Ala Glu SerGly Ser Leu Ala Val Ser Val Ser Lys Ile Val Val His 65 70 75 80 Lys AspTrp Asn Ser Asn Gln Val Ser Lys Gly Asn Asp Ile Ala Leu 85 90 95 Leu LysLeu Ala Asn Pro Val Ser Leu Thr Asp Lys Ile Gln Leu Ala 100 105 110 CysLeu Pro Pro Ala Gly Thr Ile Leu Pro Asn Asn Tyr Pro Cys Tyr 115 120 125Val Thr Gly Trp Gly Arg Leu Gln Thr Asn Gly Ala Leu Pro Asp Asp 130 135140 Leu Lys Gln Gly Arg Leu Leu Val Val Asp Tyr Ala Thr Cys Ser Ser 145150 155 160 Ser Gly Trp Trp Gly Ser Thr Val Lys Thr Asn Met Ile Cys AlaGly 165 170 175 Gly Asp Gly Val Ile Cys Thr Cys Asn Gly Asp Ser Gly GlyPro Leu 180 185 190 Asn Cys Gln Ala Ser Asp Gly Arg Trp Glu Val His GlyIle Gly Ser 195 200 205 Leu Thr Ser Val Leu Gly Cys Asn Tyr Tyr Tyr LysPro Ser Ile Phe 210 215 220 Thr Arg Val Ser Asn Tyr Asn Asp Trp Ile AsnSer Val Ile Ala Asn 225 230 235 240 Asn 239 amino acids amino acidsingle linear None 61 Val Val Gly Gly Glu Asp Ala Arg Pro His Ser TrpPro Trp Gln Ile 1 5 10 15 Ser Leu Gln Tyr Leu Lys Asn Asp Thr Trp ArgHis Thr Cys Gly Gly 20 25 30 Thr Leu Ile Ala Ser Asn Phe Val Leu Thr AlaAla His Cys Ile Ser 35 40 45 Asn Thr Xaa Thr Tyr Arg Val Ala Val Gly LysAsn Asn Leu Glu Val 50 55 60 Glu Asp Glu Glu Gly Ser Leu Phe Val Gly ValAsp Thr Ile His Val 65 70 75 80 His Lys Arg Trp Asn Ala Leu Leu Leu ArgAsn Asp Ile Ala Leu Ile 85 90 95 Lys Leu Ala Glu His Val Glu Leu Ser AspThr Ile Gln Val Ala Cys 100 105 110 Leu Pro Glu Lys Asp Ser Leu Leu ProLys Asp Tyr Pro Cys Tyr Val 115 120 125 Thr Gly Trp Gly Arg Leu Trp ThrAsn Gly Pro Ile Ala Asp Lys Leu 130 135 140 Gln Gln Gly Leu Gln Pro ValVal Asp His Ala Thr Cys Ser Arg Ile 145 150 155 160 Asp Trp Trp Gly PheArg Val Lys Lys Thr Met Val Cys Ala Gly Gly 165 170 175 Asp Gly Val IleSer Ala Cys Asn Gly Asp Ser Gly Gly Pro Leu Asn 180 185 190 Cys Gln LeuGlu Asn Gly Ser Trp Glu Val Phe Gly Ile Val Ser Phe 195 200 205 Gly SerArg Arg Gly Cys Asn Thr Arg Lys Lys Pro Val Val Tyr Thr 210 215 220 ArgVal Ser Ala Tyr Ile Asp Trp Ile Asn Glu Lys Met Gln Leu 225 230 235 230amino acids amino acid single linear None 62 Ile Val Asn Gly Glu Asp AlaVal Pro Gly Ser Trp Pro Trp Gln Val 1 5 10 15 Ser Leu Gln Asp Lys ThrGly Phe His Phe Cys Gly Gly Ser Leu Ile 20 25 30 Ser Glu Asp Trp Val ValThr Ala Ala His Cys Gly Val Arg Thr Ser 35 40 45 Asp Val Val Val Ala GlyGlu Phe Asp Gln Gly Ser Asp Glu Glu Asn 50 55 60 Ile Gln Val Leu Lys IleAla Lys Val Phe Lys Asn Pro Lys Phe Ser 65 70 75 80 Ile Leu Thr Val AsnAsn Asp Ile Thr Leu Leu Lys Leu Ala Thr Pro 85 90 95 Ala Arg Phe Ser GlnThr Val Ser Ala Val Cys Leu Pro Ser Ala Asp 100 105 110 Asp Asp Phe ProAla Gly Thr Leu Cys Ala Thr Thr Gly Trp Gly Lys 115 120 125 Thr Lys TyrAsn Ala Asn Lys Thr Pro Asp Lys Leu Gln Gln Ala Ala 130 135 140 Leu ProLeu Leu Ser Asn Ala Glu Cys Lys Lys Ser Trp Gly Arg Arg 145 150 155 160Ile Thr Asp Val Met Ile Cys Ala Gly Ala Ser Gly Val Ser Ser Cys 165 170175 Met Gly Asp Ser Gly Gly Pro Leu Val Cys Gln Lys Asp Gly Ala Trp 180185 190 Thr Leu Val Gly Ile Val Ser Trp Gly Ser Asp Thr Cys Ser Thr Ser195 200 205 Ser Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp ValGln 210 215 220 Lys Ile Leu Ala Ala Asn 225 230 248 amino acids aminoacid single linear None 63 Ile Val Gly Gly Thr Asn Ser Ser Trp Gly GluTrp Pro Trp Gln Val 1 5 10 15 Ser Leu Gln Val Lys Leu Thr Ala Gln ArgHis Leu Cys Gly Gly Ser 20 25 30 Leu Ile Gly His Gln Trp Val Leu Thr AlaAla His Cys Phe Asp Gly 35 40 45 Leu Pro Leu Gln Asp Val Trp Arg Ile TyrSer Gly Ile Leu Asn Leu 50 55 60 Ser Asp Ile Thr Lys Asp Thr Pro Phe SerGln Ile Lys Glu Ile Ile 65 70 75 80 Ile His Gln Asn Tyr Lys Val Ser GluGly Asn His Asp Ile Ala Leu 85 90 95 Ile Lys Leu Gln Ala Pro Leu Asn TyrThr Glu Phe Gln Lys Pro Ile 100 105 110 Cys Leu Pro Ser Lys Gly Asp ThrSer Thr Ile Tyr Thr Asn Cys Trp 115 120 125 Val Thr Gly Trp Gly Phe SerLys Glu Lys Gly Glu Ile Gln Asn Ile 130 135 140 Leu Gln Lys Val Asn IlePro Leu Val Thr Asn Glu Glu Cys Gln Lys 145 150 155 160 Arg Tyr Gln AspTyr Lys Ile Thr Gln Arg Met Val Cys Ala Gly Tyr 165 170 175 Lys Glu GlyGly Lys Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu 180 185 190 Val CysLys His Asn Gly Met Trp Arg Leu Val Gly Ile Thr Ser Trp 195 200 205 GlyGlu Gly Cys Ala Arg Arg Glu Gln Pro Gly Val Tyr Thr Lys Val 210 215 220Ala Glu Tyr Met Asp Trp Ile Leu Glu Lys Thr Gln Ser Ser Asp Gly 225 230235 240 Lys Ala Gln Met Gln Ser Pro Ala 245 238 amino acids amino acidsingle linear None 64 Ile Val Gly Gly Thr Ala Ser Val Arg Gly Glu TrpPro Trp Gln Val 1 5 10 15 Thr Leu His Thr Thr Ser Pro Thr Gln Arg HisLeu Cys Gly Gly Ser 20 25 30 Ile Ile Gly Asn Gln Trp Ile Leu Thr Ala AlaHis Cys Phe Tyr Gly 35 40 45 Val Glu Ser Pro Lys Ile Leu Arg Val Tyr SerGly Ile Leu Asn Gln 50 55 60 Ser Glu Ile Lys Glu Asp Thr Ser Phe Phe GlyVal Gln Glu Ile Ile 65 70 75 80 Ile His Asp Gln Tyr Lys Met Ala Glu SerGly Tyr Asp Ile Ala Leu 85 90 95 Leu Lys Leu Glu Thr Thr Val Asn Tyr ThrAsp Ser Gln Arg Pro Ile 100 105 110 Cys Leu Pro Ser Lys Gly Asp Arg AsnVal Ile Tyr Thr Asp Cys Trp 115 120 125 Val Thr Gly Trp Gly Tyr Arg LysLeu Arg Asp Lys Ile Gln Asn Thr 130 135 140 Leu Gln Lys Ala Lys Ile ProLeu Val Thr Asn Glu Glu Cys Gln Lys 145 150 155 160 Arg Tyr Arg Gly HisLys Ile Thr His Lys Met Ile Cys Ala Gly Tyr 165 170 175 Arg Glu Gly GlyLys Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu 180 185 190 Ser Cys LysHis Asn Glu Val Trp His Leu Val Gly Ile Thr Ser Trp 195 200 205 Gly GluGly Cys Ala Gln Arg Glu Arg Pro Gly Val Tyr Thr Asn Val 210 215 220 ValGlu Tyr Val Asp Trp Ile Leu Glu Lys Thr Gln Ala Val 225 230 235 235amino acids amino acid single linear None 65 Ile Val Gly Gly Ser Asn AlaLys Glu Gly Ala Trp Pro Trp Val Val 1 5 10 15 Gly Leu Tyr Tyr Gly GlyArg Leu Leu Cys Gly Ala Ser Leu Val Ser 20 25 30 Ser Asp Trp Leu Val SerAla Ala His Cys Val Tyr Gly Arg Asn Leu 35 40 45 Glu Pro Ser Lys Trp ThrAla Ile Leu Gly Leu His Met Lys Ser Asn 50 55 60 Leu Thr Ser Pro Gln ThrVal Pro Arg Leu Ile Asp Glu Ile Val Ile 65 70 75 80 Asn Pro His Tyr AsnArg Arg Arg Lys Asp Asn Asp Ile Ala Met Met 85 90 95 His Leu Glu Phe LysVal Asn Tyr Thr Asp Tyr Ile Gln Pro Ile Cys 100 105 110 Leu Pro Glu GluAsn Gln Val Phe Pro Pro Gly Arg Asn Cys Ser Ile 115 120 125 Ala Gly TrpGly Thr Val Val Tyr Gln Gly Thr Thr Ala Asn Ile Leu 130 135 140 Gln GluAla Asp Val Pro Leu Leu Ser Asn Glu Arg Cys Gln Gln Gln 145 150 155 160Met Pro Glu Tyr Asn Ile Thr Glu Asn Met Ile Cys Ala Gly Tyr Glu 165 170175 Glu Gly Gly Ile Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Met 180185 190 Cys Gln Glu Asn Asn Arg Trp Phe Leu Ala Gly Val Thr Ser Phe Gly195 200 205 Tyr Lys Cys Ala Leu Pro Asn Arg Pro Gly Val Tyr Ala Arg ValSer 210 215 220 Arg Phe Thr Glu Trp Ile Gln Ser Phe Leu His 225 230 235299 amino acids amino acid single linear None 66 Ile Thr Gly Gly Ser SerAla Val Ala Gly Gln Trp Pro Trp Gln Val 1 5 10 15 Ser Ile Thr Tyr GluGly Val His Val Cys Gly Gly Ser Leu Val Ser 20 25 30 Glu Gln Trp Val LeuSer Ala Ala His Cys Phe Pro Ser Glu His His 35 40 45 Lys Glu Ala Tyr GluVal Lys Leu Gly Ala His Gln Leu Asp Ser Tyr 50 55 60 Ser Glu Asp Ala LysVal Ser Thr Leu Lys Asp Ile Ile Pro His Pro 65 70 75 80 Ser Tyr Leu GlnGlu Gly Ser Gln Gly Asp Ile Ala Leu Leu Gln Leu 85 90 95 Ser Arg Pro IleThr Phe Ser Arg Tyr Ile Arg Pro Ile Cys Leu Pro 100 105 110 Ala Ala AsnAla Ser Phe Pro Asn Gly Leu His Cys Thr Val Thr Gly 115 120 125 Trp GlyHis Val Ala Pro Ser Val Ser Leu Leu Thr Pro Lys Pro Leu 130 135 140 GlnGln Leu Glu Val Pro Leu Ile Ser Arg Glu Thr Cys Asn Cys Leu 145 150 155160 Tyr Asn Ile Asp Ala Lys Pro Glu Glu Pro His Phe Val Gln Glu Asp 165170 175 Met Val Cys Ala Gly Tyr Val Glu Gly Gly Lys Asp Ala Cys Gln Gly180 185 190 Asp Ser Gly Gly Pro Leu Ser Cys Pro Val Glu Gly Leu Trp TyrLeu 195 200 205 Thr Gly Ile Val Ser Trp Gly Asp Ala Cys Gly Ala Arg AsnArg Pro 210 215 220 Gly Val Tyr Thr Leu Ala Ser Ser Tyr Ala Ser Trp IleGln Ser Lys 225 230 235 240 Val Thr Glu Leu Gln Pro Arg Val Val Pro GlnThr Gln Glu Ser Gln 245 250 255 Pro Asp Ser Asn Leu Cys Gly Ser His LeuAla Phe Ser Ser Ala Pro 260 265 270 Ala Gln Gly Leu Leu Arg Pro Ile LeuPhe Leu Pro Leu Gly Leu Ala 275 280 285 Leu Gly Leu Leu Ser Pro Trp LeuSer Glu His 290 295 255 amino acids amino acid single linear None 67 IleVal Gly Gly Arg Asp Thr Ser Leu Gly Arg Trp Pro Trp Gln Val 1 5 10 15Ser Leu Arg Tyr Asp Gly Ala His Leu Cys Gly Gly Ser Leu Leu Ser 20 25 30Gly Asp Trp Val Leu Thr Ala Ala His Cys Phe Pro Glu Arg Asn Arg 35 40 45Val Leu Ser Arg Trp Arg Val Phe Ala Gly Ala Val Ala Gln Ala Ser 50 55 60Pro His Gly Leu Gln Leu Gly Val Gln Ala Val Val Tyr His Gly Gly 65 70 7580 Tyr Leu Pro Phe Arg Asp Pro Asn Ser Glu Glu Asn Ser Asn Asp Ile 85 9095 Ala Leu Val His Leu Ser Ser Pro Leu Pro Leu Thr Glu Tyr Ile Gln 100105 110 Pro Val Cys Leu Pro Ala Ala Gly Gln Ala Leu Val Asp Gly Lys Ile115 120 125 Cys Thr Val Thr Gly Trp Gly Asn Thr Gln Tyr Tyr Gly Gln GlnAla 130 135 140 Gly Val Leu Gln Glu Ala Arg Val Pro Ile Ile Ser Asn AspVal Cys 145 150 155 160 Asn Gly Ala Asp Phe Tyr Gly Asn Gln Ile Lys ProLys Met Phe Cys 165 170 175 Ala Gly Tyr Pro Glu Gly Gly Ile Asp Ala CysGln Gly Asp Ser Gly 180 185 190 Gly Pro Phe Val Cys Glu Asp Ser Ile SerArg Thr Pro Arg Trp Arg 195 200 205 Leu Cys Gly Ile Val Ser Trp Gly ThrGly Cys Ala Leu Ala Gln Lys 210 215 220 Pro Gly Val Tyr Thr Lys Val SerAsp Phe Arg Glu Trp Ile Phe Gln 225 230 235 240 Ala Ile Lys Thr His SerGlu Ala Ser Gly Met Val Thr Gln Leu 245 250 255 250 amino acids aminoacid single linear None 68 Ile Val Gly Gly Lys Ala Ala Gln His Gly AlaTrp Pro Trp Met Val 1 5 10 15 Ser Leu Gln Ile Phe Arg Tyr Asn Ser HisArg Tyr His Thr Cys Gly 20 25 30 Gly Ser Leu Leu Asn Ser Arg Trp Val LeuThr Ala Ala His Cys Phe 35 40 45 Val Gly Lys Asn Asn Val His Asp Trp ArgLeu Val Phe Gly Ala Lys 50 55 60 Glu Ile Thr Tyr Gly Asn Asn Lys Pro ValLys Ala Pro Leu Gln Glu 65 70 75 80 Arg Tyr Val Glu Lys Ile Ile Ile HisGlu Lys Tyr Asn Ser Ala Thr 85 90 95 Glu Gly Asn Asp Ile Ala Leu Val GluIle Thr Pro Pro Ile Ser Cys 100 105 110 Gly Arg Phe Ile Gly Pro Gly CysLeu Pro His Phe Lys Ala Gly Leu 115 120 125 Pro Arg Gly Ser Gln Ser CysTrp Val Ala Gly Trp Gly Tyr Ile Glu 130 135 140 Glu Lys Pro Arg Pro SerSer Ile Leu Met Glu Ala Arg Val Asp Leu 145 150 155 160 Ile Asp Leu AspLeu Cys Asn Ser Thr Gln Trp Tyr Asn Gly Arg Val 165 170 175 Gln Pro ThrAsn Val Cys Ala Gly Tyr Pro Val Gly Lys Ile Asp Thr 180 185 190 Cys GlnGly Asp Ser Gly Gly Pro Leu Met Cys Lys Asp Ser Lys Glu 195 200 205 SerAla Tyr Val Val Val Gly Ile Thr Ser Trp Gly Val Gly Cys Ala 210 215 220Leu Ala Lys Arg Pro Gly Ile Tyr Thr Ala Thr Trp Pro Tyr Leu Asn 225 230235 240 Trp Ile Ala Ser Lys Ile Gly Ser Asn Ala 245 250 245 amino acidsamino acid single linear None 69 Ile Val Gly Gly Gln Glu Ala Pro Arg SerLys Trp Pro Trp Gln Val 1 5 10 15 Ser Leu Arg Val Arg Asp Arg Tyr TrpMet His Phe Cys Gly Gly Ser 20 25 30 Leu Ile His Pro Gln Trp Val Leu ThrAla Ala His Cys Leu Gly Pro 35 40 45 Asp Val Lys Asp Leu Ala Thr Leu ArgVal Gln Leu Arg Glu Gln His 50 55 60 Leu Tyr Tyr Gln Asp Gln Leu Leu ProVal Ser Arg Ile Ile Val His 65 70 75 80 Pro Gln Phe Tyr Ile Ile Gln ThrGly Ala Asp Ile Ala Leu Leu Glu 85 90 95 Leu Glu Glu Pro Val Asn Ile SerSer Arg Val His Thr Val Met Leu 100 105 110 Pro Pro Ala Ser Glu Thr PhePro Pro Gly Met Pro Cys Trp Val Thr 115 120 125 Gly Trp Gly Asp Val AspAsn Asp Glu Pro Leu Pro Pro Pro Phe Pro 130 135 140 Leu Lys Gln Val LysVal Pro Ile Met Glu Asn His Ile Cys Asp Ala 145 150 155 160 Lys Tyr HisLeu Gly Ala Tyr Thr Gly Asp Asp Val Arg Ile Ile Arg 165 170 175 Asp AspMet Leu Cys Ala Gly Asn Ser Gln Arg Asp Ser Cys Lys Gly 180 185 190 AspSer Gly Gly Pro Leu Val Cys Lys Val Asn Gly Thr Trp Leu Gln 195 200 205Ala Gly Val Val Ser Trp Asp Glu Gly Cys Ala Gln Pro Asn Arg Pro 210 215220 Gly Ile Tyr Thr Arg Val Thr Tyr Tyr Leu Asp Trp Ile His His Tyr 225230 235 240 Val Pro Lys Lys Pro 245 243 amino acids amino acid singlelinear None 70 Val Val Gly Gly Leu Val Ala Leu Arg Gly Ala His Pro TyrIle Ala 1 5 10 15 Ala Leu Tyr Trp Gly His Ser Phe Cys Ala Gly Ser LeuIle Ala Pro 20 25 30 Cys Trp Val Leu Thr Ala Ala His Cys Leu Gln Asp ArgPro Ala Pro 35 40 45 Glu Asp Leu Thr Val Val Leu Gly Gln Glu Arg Arg AsnHis Ser Cys 50 55 60 Glu Pro Cys Gln Thr Leu Ala Val Arg Ser Tyr Arg LeuHis Glu Ala 65 70 75 80 Phe Ser Pro Val Ser Tyr Gln His Asp Leu Ala LeuLeu Arg Leu Gln 85 90 95 Glu Asp Ala Asp Gly Ser Cys Ala Leu Leu Ser ProTyr Val Gln Pro 100 105 110 Val Cys Leu Pro Ser Gly Ala Ala Arg Pro SerGlu Thr Thr Leu Cys 115 120 125 Gln Val Ala Gly Trp Gly His Gln Phe GluGly Ala Glu Glu Tyr Ala 130 135 140 Ser Phe Leu Gln Glu Ala Gln Val ProPhe Leu Ser Leu Glu Arg Cys 145 150 155 160 Ser Ala Pro Asp Val His GlySer Ser Ile Leu Pro Gly Met Leu Cys 165 170 175 Ala Gly Phe Leu Glu GlyGly Thr Asp Ala Cys Gln Gly Asp Ser Gly 180 185 190 Gly Pro Leu Val CysGlu Asp Gln Ala Ala Glu Arg Arg Leu Thr Leu 195 200 205 Gln Gly Ile IleSer Trp Gly Ser Gly Cys Gly Asp Arg Asn Lys Pro 210 215 220 Gly Val TyrThr Asp Val Ala Tyr Tyr Leu Ala Trp Ile Arg Glu His 225 230 235 240 ThrVal Ser 248 amino acids amino acid single linear None 71 Ile Ile Gly GlySer Ser Ser Leu Pro Gly Ser His Pro Trp Leu Ala 1 5 10 15 Ala Ile TyrIle Gly Asp Ser Phe Cys Ala Gly Ser Leu Val His Thr 20 25 30 Cys Trp ValVal Ser Ala Ala His Cys Phe Ser His Ser Pro Pro Arg 35 40 45 Asp Ser ValSer Val Val Leu Gly Gln His Phe Phe Asn Arg Thr Thr 50 55 60 Asp Val ThrGln Thr Phe Gly Ile Glu Lys Tyr Ile Pro Tyr Thr Leu 65 70 75 80 Tyr SerVal Phe Asn Pro Ser Asp His Asp Leu Val Leu Ile Arg Leu 85 90 95 Lys LysLys Gly Asp Arg Cys Ala Thr Arg Ser Gln Phe Val Gln Pro 100 105 110 IleCys Leu Pro Glu Pro Gly Ser Thr Phe Pro Ala Gly His Lys Cys 115 120 125Gln Ile Ala Gly Trp Gly His Leu Asp Glu Asn Val Ser Gly Tyr Ser 130 135140 Ser Ser Leu Arg Glu Ala Leu Val Pro Leu Val Ala Asp His Lys Cys 145150 155 160 Ser Ser Pro Glu Val Tyr Gly Ala Asp Ile Ser Pro Asn Met LeuCys 165 170 175 Ala Gly Tyr Phe Asp Cys Lys Ser Asp Ala Cys Gln Gly AspSer Gly 180 185 190 Gly Pro Leu Ala Cys Glu Lys Asn Gly Val Ala Tyr LeuTyr Gly Ile 195 200 205 Ile Ser Trp Gly Asp Gly Cys Gly Arg Leu His LysPro Gly Val Tyr 210 215 220 Thr Arg Val Ala Asn Tyr Val Asp Trp Ile AsnAsp Arg Ile Arg Pro 225 230 235 240 Pro Arg Arg Leu Val Ala Pro Ser 245252 amino acids amino acid single linear None 72 Ile Lys Gly Gly Leu PheAla Asp Ile Ala Ser His Pro Trp Gln Ala 1 5 10 15 Ala Ile Phe Ala LysHis Arg Arg Ser Pro Gly Glu Arg Phe Leu Cys 20 25 30 Gly Gly Ile Leu IleSer Ser Cys Trp Ile Leu Ser Ala Ala His Cys 35 40 45 Phe Gln Glu Arg PhePro Pro His His Leu Thr Val Ile Leu Gly Arg 50 55 60 Thr Tyr Arg Val ValPro Gly Glu Glu Glu Gln Lys Phe Glu Val Glu 65 70 75 80 Lys Tyr Ile ValHis Lys Glu Phe Asp Asp Asp Thr Tyr Asp Asn Asp 85 90 95 Ile Ala Leu LeuGln Leu Lys Ser Asp Ser Ser Arg Cys Ala Gln Glu 100 105 110 Ser Ser ValVal Arg Thr Val Cys Leu Pro Pro Ala Asp Leu Gln Leu 115 120 125 Pro AspTrp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Glu Ala 130 135 140 LeuSer Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His Val Arg Leu 145 150 155160 Tyr Pro Ser Ser Arg Cys Thr Ser Gln His Leu Leu Asn Arg Thr Val 165170 175 Thr Asp Asn Met Leu Cys Ala Gly Asp Thr Arg Ser Gly Gly Pro Gln180 185 190 Ala Asn Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro LeuVal 195 200 205 Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly Ile Ile SerTrp Gly 210 215 220 Leu Gly Cys Gly Gln Lys Asp Val Pro Gly Val Tyr ThrLys Val Thr 225 230 235 240 Asn Tyr Leu Asp Trp Ile Arg Asp Asn Met ArgPro 245 250 253 amino acids amino acid single linear None 73 Ile Ile GlyGly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe Ala 1 5 10 15 Ala IleTyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val Cys Gly 20 25 30 Gly SerLeu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr His Cys Phe 35 40 45 Ile AspTyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr Leu Gly Arg Ser 50 55 60 Arg LeuAsn Ser Asn Thr Gln Gly Glu Met Lys Phe Glu Val Glu Asn 65 70 75 80 LeuIle Leu His Lys Asp Tyr Ser Ala Asp Thr Leu Ala His His Asn 85 90 95 AspIle Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala Gln 100 105 110Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn Asp 115 120125 Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu Asn 130135 140 Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr Val Val Lys145 150 155 160 Leu Ile Ser His Arg Glu Cys Gln Gln Pro His Tyr Tyr GlySer Glu 165 170 175 Val Thr Thr Lys Met Leu Cys Ala Ala Asp Pro Gln TrpLys Thr Asp 180 185 190 Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val CysSer Leu Gln Gly 195 200 205 Arg Met Thr Leu Thr Gly Ile Val Ser Trp GlyArg Gly Cys Ala Leu 210 215 220 Lys Asp Lys Pro Gly Val Tyr Thr Arg ValSer His Phe Leu Pro Trp 225 230 235 240 Ile Arg Ser His Thr Lys Glu GluAsn Gly Leu Ala Leu 245 250 244 amino acids amino acid single linearNone 74 Ile Val Gly Gly Lys Arg Ala Gln Leu Gly Asp Leu Pro Trp Gln Val1 5 10 15 Ala Ile Lys Asp Ala Ser Gly Ile Thr Cys Gly Gly Ile Tyr IleGly 20 25 30 Gly Cys Trp Ile Leu Thr Ala Ala His Cys Leu Arg Ala Ser LysThr 35 40 45 His Arg Tyr Gln Ile Trp Thr Thr Val Val Asp Trp Ile His ProAsp 50 55 60 Leu Lys Arg Ile Val Ile Glu Tyr Val Asp Arg Ile Ile Phe HisGlu 65 70 75 80 Asn Tyr Asn Ala Gly Thr Tyr Gln Asn Asp Ile Ala Leu IleGlu Met 85 90 95 Lys Lys Asp Gly Asn Lys Lys Asp Cys Glu Leu Pro Arg SerIle Pro 100 105 110 Ala Cys Val Pro Trp Ser Pro Tyr Leu Phe Gln Pro AsnAsp Thr Cys 115 120 125 Ile Val Ser Gly Trp Gly Arg Glu Lys Asp Asn GluArg Val Phe Ser 130 135 140 Leu Gln Trp Gly Glu Val Lys Leu Ile Ser AsnCys Ser Lys Phe Tyr 145 150 155 160 Gly Asn Arg Phe Tyr Glu Lys Glu MetGlu Cys Ala Gly Thr Tyr Asp 165 170 175 Gly Ser Ile Asp Ala Cys Lys GlyAsp Ser Gly Gly Pro Leu Val Cys 180 185 190 Met Asp Ala Asn Asn Val ThrTyr Val Trp Gly Val Val Ser Trp Gly 195 200 205 Glu Asn Cys Gly Lys ProGlu Phe Pro Gly Phe Tyr Thr Lys Val Ala 210 215 220 Asn Tyr Phe Asp TrpIle Ser Tyr His Val Gly Arg Pro Phe Ile Ser 225 230 235 240 Gln Tyr AsnVal 8 amino acids amino acid single linear 75 Asp Tyr Lys Asp Asp AspAsp Lys 1 5 21 amino acids amino acid single linear 76 Glu Gln Lys LeuIle Ser Glu Glu Asp Leu Asn Met His Thr Glu His 1 5 10 15 His His HisHis His 20

1-4. (Canceled).
 5. An isolated polypeptide comprising an amino acidsequence of SEQ. ID NO:
 24. 6. The polypeptide of claim 5, wherein saidpolypeptide is produced by recombinant expression in a host celltransformed with a polynucleotide encoding the polypeptide. 7-16.(Canceled).